Transgenic vero-cd4/ccr5 cell line

ABSTRACT

The present relation relates to a transgenic Vero cell line expressing CD4 and CCR5. The present invention encompasses the preparation and purification of immunogenic compositions which are formulated into the vaccines of the present invention.

INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/481,108 filed Apr. 6, 2017, now allowed, which is acontinuation-in-part application of U.S. patent application Ser. No.15/280,710 filed Sep. 29, 2016, now U.S. Pat. No. 9,925,258, whichclaims benefit of and priority to U.S. provisional patent applicationSer. No. 62/236,448 filed Oct. 2, 2015.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appin cited documents”) and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 10, 2017, isnamed 43094_02_2039_SL.txt and is 57,121 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a transgenic Vero cell line expressingCD4 and CCR5 for use to manufacture prophylactic and therapeuticvaccines.

BACKGROUND OF THE INVENTION

AIDS, or Acquired Immunodeficiency Syndrome, is caused by humanimmunodeficiency virus (HIV) and is characterized by several clinicalfeatures including wasting syndromes, central nervous systemdegeneration and profound immunosuppression that results inopportunistic infections and malignancies. HIV is a member of thelentivirus family of animal retroviruses, which include the visna virusof sheep and the bovine, feline, and simian immunodeficiency viruses(SIV). Two closely related types of HIV, designated HIV-1 and HIV-2,have been identified thus far, of which HIV-1 is by far the most commoncause of AIDS. However, HIV-2, which differs in genomic structure andantigenicity, causes a similar clinical syndrome.

An infectious HIV particle consists of two identical strands of RNA,each approximately 9.2 kb long, packaged within a core of viralproteins. This core structure is surrounded by a phospholipid bilayerenvelope derived from the host cell membrane that also includesvirally-encoded membrane proteins (Abbas et al., Cellular and MolecularImmunology, 4th edition, W.B. Saunders Company, 2000, p. 454). The HIVgenome has the characteristic 5′-LTR-Gag-Pol-Env-LTR-3′ organization ofthe retrovirus family. Long terminal repeats (LTRs) at each end of theviral genome serve as binding sites for transcriptional regulatoryproteins from the host and regulate viral integration into the hostgenome, viral gene expression, and viral replication.

The HIV genome encodes several structural proteins. The gag gene encodesstructural proteins of the nucleocapsid core and matrix. The pol geneencodes reverse transcriptase (RT), integrase (IN), and viral protease(PR) enzymes required for viral replication. The tat gene encodes aprotein that is required for elongation of viral transcripts. The revgene encodes a protein that promotes the nuclear export of incompletelyspliced or unspliced viral RNAs. The vif gene product enhances theinfectivity of viral particles. The vpr gene product promotes thenuclear import of viral DNA and regulates G2 cell cycle arrest. The vpuand nef genes encode proteins that down regulate host cell CD4expression and enhance release of virus from infected cells. The envgene encodes the viral envelope glycoprotein that is translated as a160-kilodalton (kDa) precursor (gp160) and cleaved by a cellularprotease to yield the external 120-kDa envelope glycoprotein (gp120) andthe transmembrane 41-kDa envelope glycoprotein (gp41), which arerequired for the infection of cells (Abbas, pp. 454-456). gp140 is amodified form of the Env glycoprotein, which contains the external120-kDa envelope glycoprotein portion and the extracellular part of thegp41 portion of Env and has characteristics of both gp120 and gp41. Thenef gene is conserved among primate lentiviruses and is one of the firstviral genes that is transcribed following infection. In vitro, severalfunctions have been described, including down-regulation of CD4 and MHCclass I surface expression, altered T-cell signaling and activation, andenhanced viral infectivity.

HIV infection initiates with gp120 on the viral particle binding to theCD4 and chemokine receptor molecules (e.g., CXCR4, CCR5) on the cellmembrane of target cells such as CD4⁺ T-cells, macrophages and dendriticcells. The bound virus fuses with the target cell and reversetranscribes the RNA genome. The resulting viral DNA integrates into thecellular genome, where it directs the production of new viral RNA, andthereby viral proteins and new virions. These virions bud from theinfected cell membrane and establish productive infections in othercells. This process also kills the originally infected cell. HIV canalso kill cells indirectly because the CD4 receptor on uninfectedT-cells has a strong affinity for gp120 expressed on the surface ofinfected cells. In this case, the uninfected cells bind, via the CD4receptor-gp120 interaction, to infected cells and fuse to form asyncytium, which cannot survive. Destruction of CD4⁺ T-lymphocytes,which are critical to immune defense, is a major cause of theprogressive immune dysfunction that is the hallmark of AIDS diseaseprogression. The loss of CD4⁺ T cells seriously impairs the body'sability to fight most invaders, but it has a particularly severe impacton the defenses against viruses, fungi, parasites and certain bacteria,including mycobacteria.

Research on the Env glycoprotein has shown that the virus has manyeffective protective mechanisms with few vulnerabilities (Wyatt &Sodroski, Science. 1998 Jun. 19; 280(5371):1884-8). For fusion with itstarget cells, HIV-1 uses a trimeric Env complex containing gp120 andgp41 subunits (Burton et al., Nat Immunol. 2004 March; 5(3):233-6). Thefusion potential of the Env complex is triggered by engagement of theCD4 receptor and a coreceptor, usually CCR5 or CXCR4. Neutralizingantibodies seem to work either by binding to the mature trimer on thevirion surface and preventing initial receptor engagement events, or bybinding after virion attachment and inhibiting the fusion process(Parren & Burton, Adv Immunol. 2001; 77:195-262). In the latter case,neutralizing antibodies may bind to epitopes whose exposure is enhancedor triggered by receptor binding. However, given the potential antiviraleffects of neutralizing antibodies, it is not unexpected that HIV-1 hasevolved multiple mechanisms to protect it from antibody binding (Johnson& Desrosiers, Annu Rev Med. 2002; 53:499-518).

There remains a need to express immunogens that elicit broadlyneutralizing antibodies. Strategies include producing molecules thatmimic the mature trimer on the virion surface, producing Env moleculesengineered to better present neutralizing antibody epitopes thanwild-type molecules, generating stable intermediates of the entryprocess to expose conserved epitopes to which antibodies could gainaccess during entry and producing epitope mimics of the broadlyneutralizing monoclonal antibodies determined from structural studies ofthe antibody-antigen complexes (Burton et al., Nat Immunol. 2004 March;5(3):233-6). However, none of these approaches have yet efficientlyelicited neutralizing antibodies with broad specificity.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentapplication.

SUMMARY OF THE INVENTION

The present invention relates to a transgenic Vero cell line thatexpress CD4/CCR5. In an advantageous embodiment, the CD4/CCR5 is derivedfrom either human or rhesus macaque. In an advantageous embodiment, thetransgenic Vero-CD4/CCR5 cell lines support Env-dependent infection andreplication by VSV- and CDV-Env chimeras, wherein the Env expressed byinfected cells comprises a native conformation and antigenicity. Theinvention encompasses manufacturing of replicating viral vectored HIVvaccines that express functional Env immunogens. Because Vero is aFDA-approved cell substrate for human vaccine production, the transgenicVero-CD4/CCR5 cell line is suitable for manufacturing human vaccines.

Transgenic Vero-CD4/CCR5 cells are useful for HIV vaccine productionsince many safety risks associated with cell substrates have beenaddressed for the Vero cell background. The unique CD4/CCR5 transgenedesign directs expression of a CCR5 and CD4 polyprotein linked by a 2Asequence (de Felipe P, Luke G A, Hughes L E, Gani D, Halpin C, Ryan M D.E unum pluribus: multiple proteins from a self-processing polyprotein.Trends Biotechnol. 2006; 24(2):68-75) that is subsequently self-cleavedresulting in 1 to 1 ratio of CD4 and CCR5 molecules.

The transgenic Vero-CD4/CCR5 cell lines are useful for producingreplicating viral vectors expressing HIV or SIV Env. Their use can alsobe expanded for use in assays requiring cells expressing CD4 and CCR5.

As the expression cassette proved effective with CD4 and CCR5, it isuseful for making cell lines expressing other polypeptides.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIGS. 1A-1C. Recombinant VSVΔG-Env. A) A linear map of the VSV genome,which contains 5 gene regions encoding the Nucleocapsid protein (N), thePhosphoprotein (P) RNA-dependent RNA polymerase subunit, Matrix protein(M), Glycoprotein (G), and the catalytic subunit of the polymerase(Large protein or L). The 11-kb RNA genome is single-stranded,nonsegmented, and negative-sense. A single promoter at the 3′ endcontrols mRNA synthesis. Transcription initiated at the 3′ endterminates and reinitiates at each gene boundary. Because reinitiationis not 100% efficient, gene regions distal to the promoter aretranscribed less efficiently generating a protein expression gradient.Changing the gene order, particularly when N is placed downstream,attenuates virus replication. A schematic of the VSV particle is shownext to the genome map. B) Genome of the VSVΔG-Env.BG505 chimera inwhich the G gene is replaced with sequence encoding HIV Env.BG505. C)Map of the VSVΔG-Env genomic clone. The VSV (Indiana serotype) genomicsequence was derived from a lab-adapted virus. The Env.BG505 genesequence is optimized to reflect VSV codon usage and relatively A+T-richnucleotide content. To support rescue of recombinant virus, the T7bacteriophage promoter is positioned to transcribe a positive-sensegenome copy precursor and subsequent cleavage by cis-acting ribozymesgenerate precise termini.

FIGS. 2A-2D. Summary of VSVΔG-Env.BG505 rescue and vaccine preparation.Steps in the process are summarized along with virus particleillustrations that show glycoprotein composition at different stages.Chimeric virus rescue is initiated by electroporating DNA (A) into Veroor VeroCD4/CCR5 cells. Virus that buds from electroporated cells then isexpanded using VeroCD4/CCR5 cells before conducting 3 rounds of clonalisolation (B). Clonal isolates are characterized, and candidates areselected for seed virus amplification and storage. Conducting thesesteps using CD4+/CCR5+ ensures that the vector is genetically stable andwill propagate efficiently using Env. When a pseudotyped vaccinepreparation is produced (C), virus infection is performed usingVeroCD4/CCR5 cells electroporated with DNA encoding G. Replication invivo (D) produces virus particles that lack the G glycoprotein.

FIGS. 3A-3B. Improvement of Env spike surface expression. A) Flowcytometry conducted with transfected 293T cells expressing modifiedEnvs. Monoclonal bnAbs used for detection are indicated at the right.Notably, antibodies PG16, PGT151, and PGT145 preferentially react withepitopes that are formed by well-ordered trimers. A linear illustrationof an Env monomer and corresponding Env-G hybrids (B) is shown below theflow cytometry data in Part A. SP, signal peptide, which is cleavedduring translational processing; TM, transmembrane; CT, cytoplasmictail; MPER, the Env membrane proximal external region; G stem; membraneproximal external region of G.

FIGS. 4A-4C. Antigenicity of Env.BG505 trimers delivered withVSVΔG-Env.BG505. Chimeric virus particles containing Env.BG505 only (A)were used for analyses in panels C-D. In B, infected VeroCD4/CCR5 wereanalyzed by flow cytometry using antibodies listed on the X-axis. In C,purified virus was adsorbed to alum after which the alum-virus complexeswere reacted with mAbs and analyzed by flow cytometry. The asterisks inB and C highlight antibodies that preferentially recognized well ordertrimers.

FIGS. 5A-5C. Immunogenicity of a prototype VSVΔG-SIV-GagEnv chimera inrhesus macaques. A) Genome map of the VSVΔG-SIV-GagEnv chimera, whichcontains the SIV Gag and Env genes. The C-terminus of Env was truncatedleaving a six-amino acid cytoplasmic domain. This was required toimprove express of SIV Env by the VSV vector. B) Three groups of animals(3 per group) were vaccinated with VSVΔG-SIV-GagEnv, VSVΔG-SIV-GagEnvprepared with a G pseudotype, or a negative control, which was liverecombinant VSV. Animals were vaccinated twice (0 and 6 weeks) with2×10⁸ pfus. Live virus in buffered solution was administered in dropsapplied to the nasal and oral cavities (1×10⁸ pfu per site). C) Anti-SIVEnv serum antibody titers were quantified by bioplex assay. To the rightside of the chart, the peak antibody titer elicited in an earlier studywith a DNA-SIV Env prime (electroporation) and Ad5-SIV Env boost isindicated with a dotted line for comparison. Assay background issubtracted from the data presented in the graph.

FIGS. 6A-6C. Genome maps comparing VSVΔG-Env.BG505 and an alternativevector design, VSV-G6-Env.BG505. A) The VSVΔG-Env.BG505 chimera genomecontains 5 genes with Env.BG505 coding sequence inserted in place of G(position 4, also see FIG. 1). Gene expression declines with increasingdistance from the transcriptional promoter located at the left end(yellow arrow, and see FIG. 1). The VSVΔG-Env.BG505 particle isillustrated with only Env incorporated on the surface, which isrepresentative of the progeny virus particles that will be produced asthe vector replicates in the vaccinee. B) In the VSV-G6-EnvBG505 vector,the G gene was reintroduced, but placed in position 6, whichdown-regulates G expression and enables stable coexpression of bothglycoproteins. C) VSV schematic for comparison to the two vectorsillustrated above.

FIG. 7. Preclinical efficacy study design. Three groups of 10 Indianrhesus macaques were vaccinated according to the timeline at the top,which shows months and weeks. The three vaccine groups included:VSVΔG-Env.BG505 chimeria, VSV-G6-Env.BG505, and saline control.Vaccination and repeated rectal challenge time points are illustrated byfilled triangles. Challenge was conducted with a heterologous clade BSHIV (SHIV SF162p3).

FIG. 8. Serum antibodies elicited by vaccination. Antibody binding toEnv.BG505 gp120 was quantified by ELISA over the course of thevaccination phase.

FIG. 9. Monitoring SHIV genome copies in the blood of infected animals.SHIV genome copies in blood samples were quantified by real-timequantitative PCR (RT-qPCR). Genome copies per ml of plasma are plottedfrom the time infection was first detected by a qPCR signal of ≥200genome copies per ml. Animal identifiers are located to the right of thegraphs. Bold indicates infected animals through the 10th challenge.

FIG. 10. SHIV infection rate during repetitive rectal challenge. Asdescribed in FIG. 7, three groups of 10 animals were vaccinated withVSVΔG-Env.BG505 (red line), VSV-G6-Env.BG505 (blue line), or saline(black line). About 5 months after the third vaccination at week 48,challenge commenced using 2.2×10⁴ TCID50 per rectal inoculation (TCID50:tissue culture infectious dose required to produce cytopathic effect in50% of inoculated cell cultures). The graph shows the number ofuninfected animals (Y axis) per group prior to commencing the SHIVchallenge protocol. SHIV challenge 10 has been completed.

FIG. 11. Env.BG505 binding antibody titers at time of SHIV infection inanimals vaccinated with VSVΔG-Env.BG505. ELISA was conducted withEnv.BG505 gp120 bound to the plate using serum samples collected frommacaques at the time challenge commenced (week 48) and periodicallyduring the challenge protocol (FIG. 7). Animals 11 and 15 were infectedat SHIV challenge 1 and 2, respectively. Animal 16 was infected atchallenge 8, which was 19 weeks after the challenge protocol commenced.Colored arrows point to ELISA titers for animals 11, 15, and 16.

FIG. 12. Generation of VSVΔG-Env.BG505 from DNA and summary of steps toproduce a seed virus for use in vaccine manufacturing.

FIG. 13. Preparation of VSVΔG-Env.BG505 with G pseudotype. The schematicsummarizes a procedure for preparing pseudotyped VSVΔG-Env.BG505.

FIG. 14 shows that VSVΔG-Env.BG505 is cytolytic and that it formsplaques after an overnight incubation on GHOST cells.

FIG. 15 shows that three Env mutations were present in the protectiveVSVΔG-Env.BG505 vaccine. Adaptive mutations emerged in Env during vectorrescue and propagation that increased Env-dependent replication inVeroCD4/CCR5 cells (human CD4/CCR5). The substitutions are stable andwere included in vaccine vector tested in macaques. The ‘adapted Env’gene was incorporated into a new VSVDG-Env.BG505 genomic DNA clone thatallowed rescue of a recombinant virus containing these mutations.

FIGS. 16A-16B show dependence of VSVΔG-EnvG.BG505 infection on CD4 andCCR5. The virus used in this experiment contained three adaptive aminosubstitutions in Env.BG505: K169T, I307T, and W672R. (A) VERO orVERO-CD4/CCR5 cell lines were infected with 1 plaque-forming unit percell (PFU/cell) of VSVΔG-Env.BG505 or a mock control. Cytopathic effectcaused by VSVΔG-Env.BG505 infection is evident only VERO-CD4/CCR5 cells.(B) HOS cells expressing CD4, CCR5 or both were infected with 0.1PFU/cell of VSVΔG-Env.BG505 or a mock control. Cytopathic effectproduced by infection is only evident on cells expressing both CD4 andCCR5.

FIG. 17A-17B shows results from a maximal tolerated dose study conductedby intracranial injection in mice. Three VSVΔG-Env.BG505 vaccinepreparations were analyzed: VSVΔG-Env.BG505 with a New JerseyG-pseudotype, VSVΔG-Env.BG505 with an Indiana G-pseudotype andVSVΔG-Env.BG505. No adverse events were observed following intracranialinoculation with VSVΔG vectors. (A) No substantial weight loss inanimals over the 14 days apart from a small decrease at day 1. There was100% survival in 10{circumflex over ( )}4, 10{circumflex over ( )}5 and10{circumflex over ( )}6 pfu groups. (B) No mortality and no paralysis,limb weakness or loss of coordination was observed in any of the groups.

FIG. 18 depicts an evaluation of CD4 gene copy number in a VERO-CD4/CCR5preclinical cell line. VERO-CD4/CCR5 cells were thawed, 3 passages wereproduced (P1, P2 and P3), cellular genomic DNA (gDNA) was purified andRNase treated, DNA concentrations were determined by UVspectrophotometry and diluted to a stock concentration of 100 ng/ml,genetic integrity was determined by PCR/gel electrophoresis andstability was monitored by qPCR. Stability of CD4 and CCR5 genes in theVERO-CD4/CCR5 cell line is determined. Passage to passage consistency ofthe VERO-CD4/CCR5 cells was monitored. Three SYBR green qPCR assays(CD4, CCR5 and a reference gene β-glucuronidase) are designed. Absolutequantification is by a standard curve method. The stability of CD4 andCD4 copy number/cell is determined by the copy ratio of CD4 toβ-glucuronidase and likewise CCR5 stability.

FIGS. 19A-19E. VSV-HIV vectors evaluated in Indian rhesus macaques. (A)The VSV genome map is colored to correspond with proteins in the virusparticle illustration. The 11-kb single-stranded, negative-sense,nonsegmented RNA genome encodes 5 proteins: (N) Nucleocapsid;Phosphoprotein (P); Large (L) RNA-dependent RNA polymerase subunit; (M)Matrix protein; (G) Glycoprotein. A single 3′ promoter controls mRNAsynthesis, with promoter-proximal genes being transcribed morefrequently. The G gene was replaced with Env.BG505 sequence inVSVΔG-Env.BG505 (B) and VSV-G6-Env.BG505 (C), with G being reintroducedat the 5′ terminus (position 6) of the VSV-G6-Env.BG505 genome.Env.BG505 encoded by both vectors was modified (FIG. 23A) to increaseincorporation into the virus particle. (D) The surface of infected VEROor VERO-CD4/CCR5 cells was analyzed by flow cytometry using monoclonalantibodies specific for: high-manose glycans (2G12); a V3 epitopecomposed of polypeptide and glycan (PGT121); the CD4 binding site innative spikes (VRCO1 and VRCO6b) or in less compact Env species (F105and IgGb6); and, native structures formed at the interface of spikesubunits (PGT145 and PGT151). (E) Purified virus particles also wereanalyzed with the same antibodies using alum as a carrier for flowcytometry.

FIGS. 20A-20B. Preclinical efficacy study. (A) Macaques were vaccinatedthree times by applying VSVΔG-EnvG505, VSV-G6-Env.BG505, or bufferedsolution to both intranasal (1×10⁸ pfus) and intraoral mucosal surfaces(1×10⁸ pfus). Intrarectal challenge with SHIV began 5 months after thefinal vaccination (study week 48). The SHIV SF162p3 challenge stock wasprepared in macaque PBMCs and has been used in prior studies. Consensusnucleotide sequencing conducted with the challenge virus verified thatthe Env gene matched Genbank Accession KF042063. Macaques with SHIVgenome copies ≥200 per ml on two successive blood draws were consideredpositive (FIG. 26) and further challenge was ended. (B) Kaplan-Meiersurvival curves by treatment assignment. P-values are from an exactlog-rank test comparing each active treatment group to the controlgroup.

FIGS. 21A-21D. Serum antibody analysis by Western blot. Western blotmembranes were prepared using purified VSVΔG-Env.BG505 as a source ofEnv.BG505 and VSV polypeptides. The membranes were placed inmultichannel devices to allow analysis of sera from individual animals.(A) Analysis of week-43 sera from all vaccinated animals and twocontrols. Labeled above the blot are the vaccine groups, animal numbers(NHP, nonhuman primate), and the SHIV challenge when infection occurred.Underlined NHP numbers indicate an animal that became infected duringSHIV challenge. Polypeptide identities are labeled at the left side.Bands corresponding to Env gp41 were not clearly evident until afterSHIV infection (FIG. 30). (B) Sera were analyzed from week 48. Anindependent full-length blot is included in FIG. 30A with all controlanimal sera. (C) Sera was analyzed from week 62 when 5 of 10 challengeswere complete. Asterisks indicate animals infected after 5 challenges.Infected Control animal 31 did not produce an Env signal probablybecause it had a more severe progressive infection (FIG. 26) thatinterfered with humoral responses against Env and Gag (FIG. 27). (D)Analysis of sera from week 79, which was ˜1 year after the finalvaccination.

FIGS. 22A-22B. Mapping Env antibody binding regions. (A) Linear map ofEnv Constant (C1-C5) and Variable (V1-V5) domains. The map breaks atfurin cleavage site between gp120 and gp41. The transmembrane (TM)region and cytoplasmic tail (CT) are labeled in gp41. Below the map,boundaries are shown for the Env fragments fused to human serum albumin(HSA) to generate ELISA substrates. (B) Analysis of sera using captureELISA and the HSA fusion proteins shown in (A). HSA without a fused Envsequence was included as a negative control. Env gp120 and gp140 ELISAsubstrates were not fused to HSA. The data from an example experiment(absorbance at 450 nm; A450) is presented as a heat map with the scaleshown at the bottom adjacent to a scale showing the SHIV challenge wheninfection occurred.

FIGS. 23A-23F. VSV-HIV vaccine design details. (A) Both vectors expressHIV Env.BG505, which was modified to increase incorporation into the VSVparticle by replacing the signal sequence, transmembrane region (TM) andcytoplasmic tail (CT) with sequence from G (serotype Indiana; IND). (B)VSVΔG-Env.BG505 particles used for vaccination were pseudotyped with G.Vaccinating with pseudotyped VSVΔG-Env.BG505 launches a more robustinfection, because G binds ubiquitous cellular receptors allowing theinitial round of infection to be independent of Env and the limitedpopulation of CD4+/CCR5+ cells. Pseudotyped virus was prepared byconducting the final amplification of vaccine material in VERO-CD4/CCR5cells expressing G. (D-F) These schematics summarize how early stages ofVSV vector infection progresses in macaques with pseudotypedVSVΔG-Env.BG505 (B) and VSV-G6-Env.BG505 (C). Both can use G to initiateprimary infection (D), but subsequent cycles of VSVΔG-Env.BG505infection and replication are Env-dependent while VSV-G6-Env.BG505 are Gdependent. Additional information on the use of G in the vaccines isprovided in FIG. 24.

FIGS. 24A-24C. VSV G serotype exchange. Because 3 vaccinations wereplanned and anti-G antibodies were known to develop when using VSVvectors that express G like VSV-G6-Env (FIG. 19C), Applicants used a Gserotype exchange strategy to minimize potential effects of anti-Gimmunity (A) Timeline of vaccination and SHIV challenge shows how the Gcomposition in the vaccines was varied. (B) For VSVΔG-Env.BG505, G wasexchanged simply by pseudotyping with G from serotype New Jersey (NJ) orG IND. Only two different G pseudotypes were used for the threesequential vaccinations (A), because interim ELISA data showed thattransient mucosal exposure to G in the pseudotyped VSVΔG-Env.BG505particle did not elicit substantial amounts of anti-G antibodies (datanot shown). (C) For sequential vaccination with VSV-G6-Env.BG505 (B),three vectors were used that differed in their G genes. The G genes camefrom different vesiculoviruses including VSV serotypes NJ or IND, orMaraba virus.

FIG. 25. VSV-HIV shedding in the oral cavity. Samples were collected at3 days after each of the three vaccinations by swabbing the oral cavity.Samples also were collected 7 days after the first vaccination. Materialcollected on the swabs was purified using procedures described in theMethods for detecting SHIV genomes in plasma after which qPCR wasperformed with an amplicon specific for the N gene. The lower limit ofmeasurement was 50 genome copies per reaction. Low quantities ofVSVΔG-EnvG.BG505 genomes were detected at 3 days following the firstvaccination in 4 animals and in 3 macaques after the third vaccination.VSV-G6-Env.BG505 genomes were detected in swabs from 8 animals ingreater quantities after the first vaccination and in 4 macaquesfollowing the third vaccination. These results indicated thatVSVΔG-Env.BG505 shedding into the oral cavity was minimal toundetectable, while the quantity of VSV-G6-Env.BG505 genomes indicatedthat some virus shedding occurred although it remains to be determinedwhether live virus was present.

FIG. 26. SHIV infection and virus loads. Blood was collected at one andtwo weeks following each challenge to assess virus loads as described inthe Methods. The plots show SHIV genome copies per ml of plasma asmeasured by RT-qPCR using a SIV Gag-specific amplicon. Animals with ≥200copies on two successive blood draws were considered positive afterwhich challenge was stopped. Animal numbers are shown to the right ofthe plots, and those positive for the Mamu-A*01 or Mamu-A*02 MHC allelesare indicated. Each group had two animals that were positive forMamu-A*01 and two positive Mamu-A*02, which have been associated withcontrol of disease progression. No macaques were included in the studywith Mamu-B*17 or Mamu-B*08 alleles associated with strong replicationcontrol. Two animals in the Control group (indicated with X) experiencedrapid disease progression and were euthanized before the end of thestudy.

FIGS. 27A-27C. Analysis of anti-Gag response to SHIV infection byWestern blotting. (A) SHIV challenge timeline and labeling key for theblots below. (B-C) Gag-specific serum antibodies were detected byreactivity with recombinant SIV Gag (SIVmac239, p55 Gag; ProteinSciences Corp.) on Western blot membranes. Week-62 serum (B) wascollected after completing the first 5 challenges and week-79 serum (C)was collected two months after the 10th challenge. Animal numbers areindicated above each lane, and in (B), an asterisk indicates thatmacaques were infected by the fifth challenge. Two infected animals inthe control group (31 and 40) did not have strong anti-Gag or anti-Env(FIG. 30C) antibody responses, which was due to rapid diseaseprogression (FIG. 26) inhibiting development of antibodies.

FIGS. 28A-28B. More detailed presentation of the SHIV infectiontimeline. (A) The Table supplements the survival curve shown in FIG. 20Band the antibody analysis in 3B by provides the timing of SHIV infectionfor each animal. (B) Boxplots showing ELISA titers during vaccinationand challenge phases. The boxplots highlight the low titers in animals11, 15, and 16 in the VSVΔG-Env.BG505 group prior to SHIV infection.Boxes show median and quartiles with whiskers extending at most 1.5times the interquartile range.

FIG. 29. CD4 and CD8 T cell frequencies in peripheral blood. PBMCsharvested two weeks after the third vaccination (week 31) werestimulated with peptides representing Env.BG505 gp120, gp41, or VSV Nafter which intracellular cytokine staining and flow cytometry wasconducted to quantify CD4 and CD8 T cells. Overall, the Env-specific Tcell frequencies in peripheral blood were low (measurable limit set at0.05%). In the VSV-G6-Env.BG505 group 5 of 10 animals were positive forgp120-specific CD4 T cells secreting IFNγ. VSV N-specific CD8 T cellsalso were detected secreting IFNγ in 9 of 10 macaques and TNFα in 4 or10 animals. Notably, the frequency of T cells in peripheral bloodspecific for Env or N were below measurable limits in the groupvaccinated with VSVΔG-Env.BG505.

FIGS. 30A-30C. Additional characterization of serum antibodies byWestern blot. Assays were performed as in FIG. 21. (A) An independentweek-48 Western blot, like the one in FIG. 21B, is shown with allcontrol animals included. The full-length blot also shows reactivitywith VSV polypeptides as described in FIG. 21A. Sera from controlanimals lacked significant VSV and Env signals as expected, except foroccasional detection of bands that migrated at positions consistent withVSV M and P. (B) A Western blot performed with sera collected at studyweek 16, which was 12 weeks after the second vaccination. The resultshowed that the Env signal was detectable at this earlier time eventhough the ELISA titers were considerably lower in the VSVΔG-Env.BG505group after the second vaccination. (C) An independent week-79 Westernblot similar to the one in FIG. 21D. The full length blot shows thatserum from infected macaques was able to detect gp41. Most controlmacaques also developed antibodies that bound gp41 except for twoanimals that had had progressive infections (macaques 31 and 40; FIG.26) and uninfected animal 32. Animal 31 was euthanized prior to thistime point.

FIG. 31. Cross clade Env binding detected with Western blot. A Westernblot assay was performed using three different VSVΔG-Env.BG505 chimerasas the source of proteins on the blot. These included clade A.BG505(different blot than in FIG. 4B), B.SF162.p3, and C.CH505 (week 100).The assay was performed as described in FIG. 21 using sera from theVSVΔG-Env.BG505 group (week 48). Animal numbers are at the top of theblot. Underlined animals were not protected during SHIV challenge.

FIG. 32. Analysis of serum antibody binding to different Env regions byWestern blotting. Sera from week 48 was analyzed as described in FIG. 21except that recombinant gp120, gp140, and HSA fusion proteins (FIG. 22A)were used as substrates. The positive control lane (+) included anti-HISantibody.

FIGS. 33A-33I. Sequence annotation of VERO-hCD4/CCR5 gene. A restrictionmap of the Vert construct shows restriction enzymes cutting a maximum oftwo times, using RELibrary as a restriction enzyme library. FIGS.33A-33I disclose the top nucleic acid sequence as SEQ ID NO: 7 and thecoded protein sequence as SEQ ID NO: 8.

FIG. 34. Gene design: VERO-CD4/CCR5 Cell Line (VERT). VERO-hCD4/CCR5:Transgenic Vero cells expression human CD4 and CCR5 receptors. sVERT3:Transgenic Vero cells expression simian CD4 and CCR5 receptors.

FIG. 35. VERO-hCD4/CCR5 clone 4F11 resembles the Vero cell.

FIG. 36. VERO-hCD4/CCR5 cytopathic effect when infected by VSV chimera.

FIG. 37. VERO-hCD4/CCR5 maintains infectivity over 20 passages.

FIG. 38. VERO-hCD4/CCR5 maintains the receptor expression over 20passages.

DETAILED DESCRIPTION

The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer may be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

As used herein, the terms “antigen” or “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The term alsorefers to proteins that are immunologically active in the sense thatonce administered to a subject (either directly or by administering tothe subject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein.

The term “antibody” includes intact molecules as well as fragmentsthereof, such as Fab, F(ab′)2, Fv and scFv which are capable of bindingthe epitope determinant. These antibody fragments retain some ability toselectively bind with its antigen or receptor and include, for example:

-   -   (i) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule can be produced        by digestion of whole antibody with the enzyme papain to yield        an intact light chain and a portion of one heavy chain;    -   (ii) Fab′, the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the 20 heavy        chain; two Fab′ fragments are obtained per antibody molecule;    -   (iii) F(ab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (iv) scFv, including a genetically engineered fragment        containing the variable region of a heavy and a light chain as a        fused single chain molecule.

General methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), which is incorporated herein byreference).

It should be understood that the proteins, including the proteins of theinvention may differ from the exact sequences illustrated and describedherein. Thus, the invention contemplates deletions, additions andsubstitutions to the sequences shown, so long as the sequences functionin accordance with the methods of the invention. In this regard,particularly preferred substitutions will generally be conservative innature, i.e., those substitutions that take place within a family ofamino acids. For example, amino acids are generally divided into fourfamilies: (1) acidic—aspartate and glutamate; (2) basic—lysine,arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar—glycine, asparagine, glutamine, cysteine, serine threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified as aromatic amino acids. It is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, or viceversa; an aspartate with a glutamate or vice versa; a threonine with aserine or vice versa; or a similar conservative replacement of an aminoacid with a structurally related amino acid, will not have a majoreffect on the biological activity. Proteins having substantially thesame amino acid sequence as the sequences illustrated and described butpossessing minor amino acid substitutions that do not substantiallyaffect the immunogenicity of the protein are, therefore, within thescope of the invention.

As used herein the terms “nucleotide sequences” and “nucleic acidsequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) sequences, including, without limitation, messenger RNA (mRNA),DNA/RNA hybrids, or synthetic nucleic acids. The nucleic acid can besingle-stranded, or partially or completely double-stranded (duplex).Duplex nucleic acids can be homoduplex or heteroduplex.

As used herein the term “transgene” may be used to refer to“recombinant” nucleotide sequences that may be derived from any of thenucleotide sequences encoding the proteins of the present invention. Theterm “recombinant” means a nucleotide sequence that has been manipulated“by man” and which does not occur in nature, or is linked to anothernucleotide sequence or found in a different arrangement in nature. It isunderstood that manipulated “by man” means manipulated by someartificial means, including by use of machines, codon optimization,restriction enzymes, etc.

For example, in one embodiment the nucleotide sequences may be mutatedsuch that the activity of the encoded proteins in vivo is abrogated. Forexample, each of the Gag, Pol, Env, Nef, RT, and Int sequences of theinvention may be altered in these ways.

As regards codon optimization, the nucleic acid molecules of theinvention have a nucleotide sequence that encodes the antigens of theinvention and can be designed to employ codons that are used in thegenes of the subject in which the antigen is to be produced. Manyviruses, including HIV and other lentiviruses, use a large number ofrare codons and, by altering these codons to correspond to codonscommonly used in the desired subject, enhanced expression of theantigens can be achieved. In a preferred embodiment, the codons used are“humanized” codons, i.e., the codons are those that appear frequently inhighly expressed human genes (Andre et al., J. Virol. 72:1497-1503,1998) instead of those codons that are frequently used by HIV. Suchcodon usage provides for efficient expression of the transgenic HIVproteins in human cells. Any suitable method of codon optimization maybe used. Such methods, and the selection of such methods, are well knownto those of skill in the art. In addition, there are several companiesthat will optimize codons of sequences, such as Geneart (geneart.com).Thus, the nucleotide sequences of the invention can readily be codonoptimized.

Advantageously, Applicants codon optimize the Env gene so it has thecodon bias that is characteristic of VSV. This also results in arelatively low Guanine+Cytosine content of about 40-45%. See, e.g.,Rabinovich et al., PLoS One. 2014 Sep. 12; 9(9):e106597. doi:10.1371/journal.pone.0106597. eCollection 2014.

The invention further encompasses nucleotide sequences encodingfunctionally and/or antigenically equivalent variants and derivatives ofthe antigens of the invention and functionally equivalent fragmentsthereof. These functionally equivalent variants, derivatives, andfragments display the ability to retain antigenic activity. Forinstance, changes in a DNA sequence that do not change the encoded aminoacid sequence, as well as those that result in conservativesubstitutions of amino acid residues, one or a few amino acid deletionsor additions, and substitution of amino acid residues by amino acidanalogs are those which will not significantly affect properties of theencoded polypeptide. Conservative amino acid substitutions areglycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan. In oneembodiment, the variants have at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% homology oridentity to the antigen, epitope, immunogen, peptide or polypeptide ofinterest.

For the purposes of the present invention, sequence identity or homologyis determined by comparing the sequences when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. In particular,sequence identity may be determined using any of a number ofmathematical algorithms. A nonlimiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268,modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448.

Advantageous for use according to the present invention is the WU-BLAST(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0executable programs for several UNIX platforms can be downloaded fromftp://blast.wustl.edu/blast/executables. This program is based onWU-BLAST version 1.4, which in turn is based on the public domainNCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignmentstatistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschulet al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States,1993; Nature Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl.Acad. Sci. USA 90: 5873-5877; all of which are incorporated by referenceherein).

The various recombinant nucleotide sequences and proteins of theinvention are made using standard recombinant DNA and cloningtechniques. Such techniques are well known to those of skill in the art.See for example, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al. 1989).

The nucleotide sequences of the present invention may be inserted into“vectors.” The term “vector” is widely used and understood by those ofskill in the art, and as used herein the term “vector” is usedconsistent with its meaning to those of skill in the art. For example,the term “vector” is commonly used by those skilled in the art to referto a vehicle that allows or facilitates the transfer of nucleic acidmolecules from one environment to another or that allows or facilitatesthe manipulation of a nucleic acid molecule.

Any vector that allows expression of the proteins of the presentinvention may be used in accordance with the present invention. Incertain embodiments, the antigens and/or antibodies of the presentinvention may be used in vitro (such as using cell-free expressionsystems) and/or in cultured cells grown in vitro in order to produce theencoded antigens, such as HIV-antigens, and/or antibodies which may thenbe used for various applications such as in the production ofproteinaceous vaccines. For such applications, any vector that allowsexpression of the antigens and/or antibodies in vitro and/or in culturedcells may be used.

For applications where it is desired that the proteins be expressed invivo, for example when the transgenes of the invention are used in DNAor DNA-containing vaccines, any vector that allows for the expression ofthe proteins of the present invention and is safe for use in vivo may beused. In preferred embodiments the vectors used are safe for use inhumans, mammals and/or laboratory animals.

For the proteins of the present invention to be expressed, the proteincoding sequence should be “operably linked” to regulatory or nucleicacid control sequences that direct transcription and translation of theprotein. As used herein, a coding sequence and a nucleic acid controlsequence or promoter are said to be “operably linked” when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the coding sequence under theinfluence or control of the nucleic acid control sequence. The “nucleicacid control sequence” can be any nucleic acid element, such as, but notlimited to promoters, enhancers, IRES, introns, and other elementsdescribed herein that direct the expression of a nucleic acid sequenceor coding sequence that is operably linked thereto. The term “promoter”will be used herein to refer to a group of transcriptional controlmodules that are clustered around the initiation site for RNA polymeraseII and that when operationally linked to the protein coding sequences ofthe invention lead to the expression of the encoded protein. Theexpression of the transgenes of the present invention can be under thecontrol of a constitutive promoter or of an inducible promoter, whichinitiates transcription only when exposed to some particular externalstimulus, such as, without limitation, antibiotics such as tetracycline,hormones such as ecdysone, or heavy metals. The promoter can also bespecific to a particular cell-type, tissue or organ. Many suitablepromoters and enhancers are known in the art, and any such suitablepromoter or enhancer may be used for expression of the transgenes of theinvention. For example, suitable promoters and/or enhancers can beselected from the Eukaryotic Promoter Database (EPDB).

The present invention relates to a Vero cell line transformed with andexpressing a cluster of differentiation 4 (CD4) receptor and a C—Cchemokine receptor type 5 (CCR5 receptor).

The Vero cell lineage was isolated from kidney epithelial cellsextracted from an African green monkey (Chlorocebus sp.; formerly calledCercopithecus aethiops, this group of monkeys has been split intoseveral different species). The genome sequence was determined by OsadaN, Kohara A, Yamaji T, Hirayama N, Kasai F, Sekizuka T, Kuroda M, HanadaK (2014). “The genome landscape of the African green monkeykidney-derived Vero cell line”. DNA Research. 21: 673-83.doi:10.1093/dnares/dsu029. The ATCC supplies various different Verocells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587.Vero cells support the growth of pathogens such as: pneumoviruses, suchas RSV-A and RSV-B; human metapneumoviruses (HMPV); morbilliviruses,such as measles virus; paramyxoviruses, such as mumps virus andparainfluenza virus; rubellavirus; human and avian coronaviruses;picornaviruses, such as entroviruses, echoviruses and coxsackie viruses,and porcine SVDV and Teschen-Talfan virus; mammalian and avianreoviruses; herpesviruses, such as HSV-1 and HSV-2; simian and humanadenoviruses; varicella zoster virus (VZV); polyomaviruses, such as JC,BK and SV-40; bimaviruses, such as gumborovirus; porcine circoviruses;canine parvovirus; and Chlamydia.

CD4 is a co-receptor that assists the T cell receptor (TCR) incommunicating with an antigen-presenting cell. Using its intracellulardomain, CD4 amplifies the signal generated by the TCR by recruiting anenzyme, the tyrosine kinase Lck, which is essential for activating manymolecular components of the signaling cascade of an activated T cell.Various types of T helper cells are thereby produced. CD4 also interactsdirectly with MHC class II molecules on the surface of theantigen-presenting cell using its extracellular domain. Theextracellular domain adopts an immunoglobulin-like beta-sandwich withseven strands in 2 beta sheets, in a Greek key topology.

During antigen presentation, both the TCR complex and CD4 are recruitedto bind to different regions of the MHCII molecule (α1/β1 and β2,respectively). Close proximity between the TCR complex and CD4 in thissituation means the Lck kinase bound to the cytoplasmic tail of CD4 isable to tyrosine-phosphorylate the Immunoreceptor tyrosine activationmotifs (ITAM) present on the cytoplasmic domains of CD3. PhosphorylatedITAM motifs on CD3 recruits and activates SH2 domain-containing proteintyrosine kinases (PTK) such as Zap70 to further mediate downstreamsignal transduction via tyrosine phosphorylation, leading totranscription factor activation including NF-κB and consequent T cellactivation.

Human CD4 amino acid sequences may be found, for example, in Crise etal., J. Virol. 64:5585-5593 (1990); Lusso et al., Proc. Natl. Acad. Sci.U.S.A. 91:3872-3876 (1994); Sharma et al., Biochemistry 44:16192-16202(2005); Lindwasser et al., Curr. Mol. Med. 7:171-184 (2007) and Kwong etal., Nature 393:648-659 (1998).

Simian CD4 amino acid sequences may be found, for example, in Fomsgaardet al., Eur J Immunol. 1992 November; 22(11):2973-81.

C—C chemokine receptor type 5, also known as CCR5 or CD195, is a proteinon the surface of white blood cells that is involved in the immunesystem as it acts as a receptor for chemokines. This is the process bywhich T cells are attracted to specific tissue and organ targets. Manyforms of HIV, the virus that causes AIDS, initially use CCR5 to enterand infect host cells.

Human CD4 amino acid sequences may be found, for example, in Rizzuto etal., Science 280:1949-1953 (1998) and Schnur et al., J. MOl. Biol.410:778-797 (2011).

Simian CD4 amino acid sequences may be found, for example, in Kunstmanet al., J Virol. 2003 November; 77(22): 12310-12318.

In an advantageous embodiment, HIV-1 utilizes CD4 to gain entry intohost T-cells and achieves this through viral envelope protein gp120. Thebinding to CD4 creates a shift in the conformation of gp120 allowingHIV-1 to bind to a CCR5 or CXCR4 co-receptor expressed on the host cell.Following a structural change in viral protein gp41, HIV inserts afusion peptide into the host cell that allows the outer membrane of thevirus to fuse with the cell membrane.

The present invention relates to a recombinant vesicular stomatitisvirus (VSV) vector expressing a foreign epitope. Advantageously, theepitope is an HIV epitope. Any HIV epitope may be expressed in a VSVvector. Advantageously, the HIV epitope is an HIV antigen, HIV epitopeor an HIV immunogen, such as, but not limited to, the HIV antigens, HIVepitopes or HIV immunogens of U.S. Pat. Nos. 7,341,731; 7,335,364;7,329,807; 7,323,553; 7,320,859; 7,311,920; 7,306,798; 7,285,646;7,285,289; 7,285,271; 7,282,364; 7,273,695; 7,270,997; 7,262,270;7,244,819; 7,244,575; 7,232,567; 7,232,566; 7,223,844; 7,223,739;7,223,534; 7,223,368; 7,220,554; 7,214,530; 7,211,659; 7,211,432;7,205,159; 7,198,934; 7,195,768; 7,192,555; 7,189,826; 7,189,522;7,186,507; 7,179,645; 7,175,843; 7,172,761; 7,169,550; 7,157,083;7,153,509; 7,147,862; 7,141,550; 7,129,219; 7,122,188; 7,118,859;7,118,855; 7,118,751; 7,118,742; 7,105,655; 7,101,552; 7,097,9717,097,842; 7,094,405; 7,091,049; 7,090,648; 7,087,377; 7,083,787;7,070,787; 7,070,781; 7,060,273; 7,056,521; 7,056,519; 7,049,136;7,048,929; 7,033,593; 7,030,094; 7,022,326; 7,009,037; 7,008,622;7,001,759; 6,997,863; 6,995,008; 6,979,535; 6,974,574; 6,972,126;6,969,609; 6,964,769; 6,964,762; 6,958,158; 6,956,059; 6,953,689;6,951,648; 6,946,075; 6,927,031; 6,919,319; 6,919,318; 6,919,077;6,913,752; 6,911,315; 6,908,617; 6,908,612; 6,902,743; 6,900,010;6,893,869; 6,884,785; 6,884,435; 6,875,435; 6,867,005; 6,861,234;6,855,539; 6,841,381 6,841,345; 6,838,477; 6,821,955; 6,818,392;6,818,222; 6,815,217; 6,815,201; 6,812,026; 6,812,025; 6,812,024;6,808,923; 6,806,055; 6,803,231; 6,800,613; 6,800,288; 6,797,811;6,780,967; 6,780,598; 6,773,920; 6,764,682; 6,761,893; 6,753,015;6,750,005; 6,737,239; 6,737,067; 6,730,304; 6,720,310; 6,716,823;6,713,301; 6,713,070; 6,706,859; 6,699,722; 6,699,656; 6,696,291;6,692,745; 6,670,181; 6,670,115; 6,664,406; 6,657,055; 6,657,050;6,656,471; 6,653,066; 6,649,409; 6,649,372; 6,645,732; 6,641,816;6,635,469; 6,613,530; 6,605,427; 6,602,709 6,602,705; 6,600,023;6,596,477; 6,596,172; 6,593,103; 6,593,079; 6,579,673; 6,576,758;6,573,245; 6,573,040; 6,569,418; 6,569,340; 6,562,800; 6,558,961;6,551,828; 6,551,824; 6,548,275; 6,544,780; 6,544,752; 6,544,728;6,534,482; 6,534,312; 6,534,064; 6,531,572; 6,531,313; 6,525,179;6,525,028; 6,524,582; 6,521,449; 6,518,030; 6,518,015; 6,514,691;6,514,503; 6,511,845; 6,511,812; 6,511,801; 6,509,313; 6,506,384;6,503,882; 6,495,676; 6,495,526; 6,495,347; 6,492,123; 6,489,131;6,489,129; 6,482,614; 6,479,286; 6,479,284; 6,465,634; 6,461,6156,458,560; 6,458,527; 6,458,370; 6,451,601; 6,451,592; 6,451,323;6,436,407; 6,432,633; 6,428,970; 6,428,952; 6,428,790; 6,420,139;6,416,997; 6,410,318; 6,410,028; 6,410,014; 6,407,221; 6,406,710;6,403,092; 6,399,295; 6,392,013; 6,391,657; 6,384,198; 6,380,170;6,376,170; 6,372,426; 6,365,187; 6,358,739; 6,355,248; 6,355,247;6,348,450; 6,342,372; 6,342,228; 6,338,952; 6,337,179; 6,335,183;6,335,017; 6,331,404; 6,329,202; 6,329,173; 6,328,976; 6,322,964;6,319,666; 6,319,665; 6,319,500; 6,319,494; 6,316,205; 6,316,003;6,309,633; 6,306,625 6,296,807; 6,294,322; 6,291,239; 6,291,157;6,287,568; 6,284,456; 6,284,194; 6,274,337; 6,270,956; 6,270,769;6,268,484; 6,265,562; 6,265,149; 6,262,029; 6,261,762; 6,261,571;6,261,569; 6,258,599; 6,258,358; 6,248,332; 6,245,331; 6,242,461;6,241,986; 6,235,526; 6,235,466; 6,232,120; 6,228,361; 6,221,579;6,214,862; 6,214,804; 6,210,963; 6,210,873; 6,207,185; 6,203,974;6,197,755; 6,197,531; 6,197,496; 6,194,142; 6,190,871; 6,190,666;6,168,923; 6,156,302; 6,153,408; 6,153,393; 6,153,392; 6,153,378;6,153,377; 6,146,635; 6,146,614; 6,143,876 6,140,059; 6,140,043;6,139,746; 6,132,992; 6,124,306; 6,124,132; 6,121,006; 6,120,990;6,114,507; 6,114,143; 6,110,466; 6,107,020; 6,103,521; 6,100,234;6,099,848; 6,099,847; 6,096,291; 6,093,405; 6,090,392; 6,087,476;6,083,903; 6,080,846; 6,080,725; 6,074,650; 6,074,646; 6,070,126;6,063,905; 6,063,564; 6,060,256; 6,060,064; 6,048,530; 6,045,788;6,043,347; 6,043,248; 6,042,831; 6,037,165; 6,033,672; 6,030,772;6,030,770; 6,030,618; 6,025,141; 6,025,125; 6,020,468; 6,019,979;6,017,543; 6,017,537; 6,015,694; 6,015,661; 6,013,484; 6,013,432;6,007,838; 6,004,811; 6,004,807; 6,004,763; 5,998,132; 5,993,819;5,989,806; 5,985,926; 5,985,641; 5,985,545; 5,981,537; 5,981,505;5,981,170; 5,976,551; 5,972,339; 5,965,371; 5,962,428; 5,962,318;5,961,979; 5,961,970; 5,958,765; 5,958,422; 5,955,647; 5,955,342;5,951,986; 5,951,975; 5,942,237; 5,939,277; 5,939,074; 5,935,580;5,928,930; 5,928,913; 5,928,644; 5,928,642; 5,925,513; 5,922,550;5,922,325; 5,919,458; 5,916,806; 5,916,563; 5,914,395; 5,914,109;5,912,338; 5,912,176; 5,912,170; 5,906,936; 5,895,650; 5,891,623;5,888,726; 5,885,580 5,885,578; 5,879,685; 5,876,731; 5,876,716;5,874,226; 5,872,012; 5,871,747; 5,869,058; 5,866,694; 5,866,341;5,866,320; 5,866,319; 5,866,137; 5,861,290; 5,858,740; 5,858,647;5,858,646; 5,858,369; 5,858,368; 5,858,366; 5,856,185; 5,854,400;5,853,736; 5,853,725; 5,853,724; 5,852,186; 5,851,829; 5,851,529;5,849,475; 5,849,288; 5,843,728; 5,843,723; 5,843,640; 5,843,635;5,840,480; 5,837,510; 5,837,250; 5,837,242; 5,834,599; 5,834,441;5,834,429; 5,834,256; 5,830,876; 5,830,641; 5,830,475; 5,830,458;5,830,457; 5,827,749; 5,827,723; 5,824,497 5,824,304; 5,821,047;5,817,767; 5,817,754; 5,817,637; 5,817,470; 5,817,318; 5,814,482;5,807,707; 5,804,604; 5,804,371; 5,800,822; 5,795,955; 5,795,743;5,795,572; 5,789,388; 5,780,279; 5,780,038; 5,776,703; 5,773,260;5,770,572; 5,766,844; 5,766,842; 5,766,625; 5,763,574; 5,763,190;5,762,965; 5,759,769; 5,756,666; 5,753,258; 5,750,373; 5,747,641;5,747,526; 5,747,028; 5,736,320; 5,736,146; 5,733,760; 5,731,189;5,728,385; 5,721,095; 5,716,826; 5,716,637; 5,716,613; 5,714,374;5,709,879; 5,709,860; 5,709,843; 5,705,331; 5,703,057; 5,702,7075,698,178; 5,688,914; 5,686,078; 5,681,831; 5,679,784; 5,674,984;5,672,472; 5,667,964; 5,667,783; 5,665,536; 5,665,355; 5,660,990;5,658,745; 5,658,569; 5,643,756; 5,641,624; 5,639,854; 5,639,598;5,637,677; 5,637,455; 5,633,234; 5,629,153; 5,627,025; 5,622,705;5,614,413; 5,610,035; 5,607,831; 5,606,026; 5,601,819; 5,597,688;5,593,972; 5,591,829; 5,591,823; 5,589,466; 5,587,285; 5,585,254;5,585,250; 5,580,773; 5,580,739; 5,580,563; 5,573,916; 5,571,667;5,569,468; 5,558,865; 5,556,745; 5,550,052; 5,543,328; 5,541,100;5,541,057; 5,534,406 5,529,765; 5,523,232; 5,516,895; 5,514,541;5,510,264; 5,500,161; 5,480,967; 5,480,966; 5,470,701; 5,468,606;5,462,852; 5,459,127; 5,449,601; 5,447,838; 5,447,837; 5,439,809;5,439,792; 5,418,136; 5,399,501; 5,397,695; 5,391,479; 5,384,240;5,374,519; 5,374,518; 5,374,516; 5,364,933; 5,359,046; 5,356,772;5,354,654; 5,344,755; 5,335,673; 5,332,567; 5,320,940; 5,317,009;5,312,902; 5,304,466; 5,296,347; 5,286,852, 5,268,265; 5,264,356;5,264,342; 5,260,308; 5,256,767; 5,256,561; 5,252,556; 5,230,998;5,230,887; 5,227,159; 5,225,347; 5,221,610, 5,217,861; 5,208,321;5,206,136; 5,198,346; 5,185,147; 5,178,865; 5,173,400; 5,173,399;5,166,050; 5,156,951, 5,135,864; 5,122,446; 5,120,662; 5,103,836;5,100,777; 5,100,662; 5,093,230; 5,077,284; 5,070,010; 5,068,174;5,066,782; 5,055,391; 5,043,262; 5,039,604; 5,039,522; 5,030,718;5,030,555; 5,030,449; 5,019,387; 5,013,556; 5,008,183; 5,004,697;4,997,772; 4,983,529; 4,983,387; 4,965,069; 4,945,082; 4,921,787;4,918,166; 4,900,548; 4,888,290; 4,886,742; 4,885,235; 4,870,003;4,869,903; 4,861,707; 4,853,326; 4,839,288; 4,833,072 and 4,795,739.

The vector of the present invention advantageously encodes for anEnv.BG505 immunogen which may be encoded by a VSVΔG-Env.BG505 vaccine.The immunogen advantageously has the sequence as provided in SEQ ID NO:2.

In another embodiment, the vector of the present invention may comprisea sequence of a VSVΔG-Env.BG505 genomic clone. The genomic cloneadvantageously has the sequence as provided as SEQ ID NO: 1.

Advantageously, the HIV epitope may be an Env precursor or gp160 epitopeor immunogen. The Env precursor or gp160 epitope may be recognized byantibodies PG9, PG16, 2G12, b12, 15 2F5, 4E10, Z13, or other broadpotent neutralizing antibodies.

Adaptive mutations emerged in Env during vector rescue and propagationthat increased Env-dependent replication in VeroCD4/CCR5 cells (humanCD4/CCR5) (see, e.g., FIG. 15). The substitutions are stable andincluded in vaccine vector tested in macaques. The ‘adapted virus’ isadvanced as a genomic DNA clone containing these coding changes and hasbeen show to support rescue of recombinant virus. Therefore, the presentinvention also encompasses mutations of env that may increaseEnv-dependent replication and/or contribute to immunogenicity. TheEnv-VSV G hybrid (EnvG) mutations may include mutations of the lysine atAA position 169, the isoleucine at AA position 307 and/or the tryptophanat AA position 672. In an especially advantageous embodiment, themutations are K169T, I307T and/or W672R. Other env mutations may be atP493, M343, K168, E168, Q440 and/or L494. In an advantageous embodiment,the mutations may be M343T, K168E, E168K, E164G, Q440R and/or L494F.see, e.g., Hoffenberg et al., J. Virol. May 2013 vol. 87 no. 105372-5383 for Env sequences and alignments.

In another embodiment, HIV, or immunogenic fragments thereof, may beutilized as the HIV epitope. For example, the HIV nucleotides of U.S.Pat. Nos. 7,393,949, 7,374,877 7,306,901, 7,303,754, 7,173,014,7,122,180, 7,078,516, 7,022,814, 6,974,866, 6,958,211, 6,949,337,6,946,254, 6,896,900, 6,887,977, 6,870,045, 6,803,187, 6,794,129,6,773,915, 6,768,004, 6,706,268, 6,696,291, 6,692,955, 6,656,706,6,649,409, 6,627,442, 6,610,476, 6,602,705, 6,582,920, 6,557,296,6,531,587, 6,531,137, 6,500,623, 6,448,078, 6,429,306, 6,420,545,6,410,013, 6,407,077, 6,395,891, 6,355,789, 6,335,158, 6,323,185,6,316,183, 6,303,293, 6,300,056, 6,277,561, 6,270,975, 6,261,564,6,225,045, 6,222,024, 6,194,391, 6,194,142, 6,162,631, 6,114,167,6,114,109, 6,090,392, 6,060,587, 6,057,102, 6,054,565, 6,043,081,6,037,165, 6,034,233, 6,033,902, 6,030,769, 6,020,123, 6,015,661,6,010,895, 6,001,555, 5,985,661, 5,980,900, 5,972,596, 5,939,538,5,912,338, 5,869,339, 5,866,701, 5,866,694, 5,866,320, 5,866,137,5,864,027, 5,861,242, 5,858,785, 5,858,651, 5,849,475, 5,843,638,5,840,480, 5,821,046, 5,801,056, 5,786,177, 5,786,145, 5,773,247,5,770,703, 5,756,674, 5,741,706, 5,705,612, 5,693,752, 5,688,637,5,688,511, 5,684,147, 5,665,577, 5,585,263, 5,578,715, 5,571,712,5,567,603, 5,554,528, 5,545,726, 5,527,895, 5,527,894, 5,223,423,5,204,259, 5,144,019, 5,051,496 and 4,942,122 are useful for the presentinvention.

Any epitope recognized by an anti-HIV antibody may be used in thepresent invention. For example, the anti-HIV antibodies of U.S. Pat.Nos. 6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743, 6,534,312,6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646, 6,063,564,6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304, 5,773,247,5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009, 4,983,529,4,886,742, 4,870,003 and 4,795,739 are useful for the present invention.Furthermore, monoclonal anti-HIV antibodies of U.S. Pat. Nos. 7,074,556,7,074,554, 7,070,787, 7,060,273, 7,045,130, 7,033,593, RE39,057,7,008,622, 6,984,721, 6,972,126, 6,949,337, 6,946,465, 6,919,077,6,916,475, 6,911,315, 6,905,680, 6,900,010, 6,825,217, 6,824,975,6,818,392, 6,815,201, 6,812,026, 6,812,024, 6,797,811, 6,768,004,6,703,019, 6,689,118, 6,657,050, 6,608,179, 6,600,023, 6,596,497,6,589,748, 6,569,143, 6,548,275, 6,525,179, 6,524,582, 6,506,384,6,498,006, 6,489,131, 6,465,173, 6,461,612, 6,458,933, 6,432,633,6,410,318, 6,406,701, 6,395,275, 6,391,657, 6,391,635, 6,384,198,6,376,170, 6,372,217, 6,344,545, 6,337,181, 6,329,202, 6,319,665,6,319,500, 6,316,003, 6,312,931, 6,309,880, 6,296,807, 6,291,239,6,261,558, 6,248,514, 6,245,331, 6,242,197, 6,241,986, 6,228,361,6,221,580, 6,190,871, 6,177,253, 6,146,635, 6,146,627, 6,146,614,6,143,876, 6,132,992, 6,124,132, RE36,866, 6,114,143, 6,103,238,6,060,254, 6,039,684, 6,030,772, 6,020,468, 6,013,484, 6,008,044,5,998,132, 5,994,515, 5,993,812, 5,985,545, 5,981,278, 5,958,765,5,939,277, 5,928,930, 5,922,325, 5,919,457, 5,916,806, 5,914,109,5,911,989, 5,906,936, 5,889,158, 5,876,716, 5,874,226, 5,872,012,5,871,732, 5,866,694, 5,854,400, 5,849,583, 5,849,288, 5,840,480,5,840,305, 5,834,599, 5,831,034, 5,827,723, 5,821,047, 5,817,767,5,817,458, 5,804,440, 5,795,572, 5,783,670, 5,776,703, 5,773,225,5,766,944, 5,753,503, 5,750,373, 5,747,641, 5,736,341, 5,731,189,5,707,814, 5,702,707, 5,698,178, 5,695,927, 5,665,536, 5,658,745,5,652,138, 5,645,836, 5,635,345, 5,618,922, 5,610,035, 5,607,847,5,604,092, 5,601,819, 5,597,896, 5,597,688, 5,591,829, 5,558,865,5,514,541, 5,510,264, 5,478,753, 5,374,518, 5,374,516, 5,344,755,5,332,567, 5,300,433, 5,296,347, 5,286,852, 5,264,221, 5,260,308,5,256,561, 5,254,457, 5,230,998, 5,227,159, 5,223,408, 5,217,895,5,180,660, 5,173,399, 5,169,752, 5,166,050, 5,156,951, 5,140,105,5,135,864, 5,120,640, 5,108,904, 5,104,790, 5,049,389, 5,030,718,5,030,555, 5,004,697, 4,983,529, 4,888,290, 4,886,742 and 4,853,326, arealso useful for the present invention.

The vectors used in accordance with the present invention shouldtypically be chosen such that they contain a suitable gene regulatoryregion, such as a promoter or enhancer, such that the proteins of theinvention can be expressed.

For example, when the aim is to express the proteins of the invention invitro, or in cultured cells, or in any prokaryotic or eukaryotic systemfor the purpose of producing the protein(s), then any suitable vectorcan be used depending on the application. For example, plasmids, viralvectors, bacterial vectors, protozoal vectors, insect vectors,baculovirus expression vectors, yeast vectors, mammalian cell vectors,and the like, can be used. Suitable vectors can be selected by theskilled artisan taking into consideration the characteristics of thevector and the requirements for expressing the proteins under theidentified circumstances.

When the aim is to express the proteins of the invention in vivo in asubject, for example in order to generate an immune response against anantigen and/or protective immunity, expression vectors that are suitablefor expression on that subject, and that are safe for use in vivo,should be chosen. In an advantageous embodiment, the antigen is anHIV-antigen. For example, in some embodiments it may be desired toexpress the proteins of the invention in a laboratory animal, such asfor pre-clinical testing of the immunogenic compositions and vaccines ofthe invention. In other embodiments, it will be desirable to express theproteins of the invention in human subjects, such as in clinical trialsand for actual clinical use of the immunogenic compositions and vaccineof the invention. Any vectors that are suitable for such uses can beemployed, and it is well within the capabilities of the skilled artisanto select a suitable vector. In some embodiments it may be preferredthat the vectors used for these in vivo applications are attenuated tovector from amplifying in the subject. For example, if plasmid vectorsare used, preferably they will lack an origin of replication thatfunctions in the subject so as to enhance safety for in vivo use in thesubject. If viral vectors are used, preferably they are attenuated orreplication-defective in the subject, again, so as to enhance safety forin vivo use in the subject.

In preferred embodiments of the present invention viral vectors areused. Viral expression vectors are well known to those skilled in theart and include, for example, viruses such as adenoviruses,adeno-associated viruses (AAV), alphaviruses, herpesviruses,retroviruses and poxviruses, including avipox viruses, attenuatedpoxviruses, vaccinia viruses, and particularly, the modified vacciniaAnkara virus (MVA; ATCC Accession No. VR-1566). Such viruses, when usedas expression vectors are innately non-pathogenic in the selectedsubjects such as humans or have been modified to render themnon-pathogenic in the selected subjects. For example,replication-defective adenoviruses and alphaviruses are well known andcan be used as gene delivery vectors.

The present invention relates to recombinant vesicular stomatitis (VSV)vectors, however, other vectors may be contemplated in other embodimentsof the invention such as, but not limited to, prime boost administrationwhich may comprise administration of a recombinant VSV vector incombination with another recombinant vector expressing one or more HIVepitopes.

VSV is a very practical, safe, and immunogenic vector for conductinganimal studies, and an attractive candidate for developing vaccines foruse in humans. VSV is a member of the Rhabdoviridae family of envelopedviruses containing a nonsegmented, negative-sense RNA genome. The genomeis composed of 5 genes arranged sequentially 3′-N-P-M-G-L-5′, eachencoding a polypeptide found in mature virions. Notably, the surfaceglycoprotein G is a transmembrane polypeptide that is present in theviral envelope as a homotrimer, and like Env, it mediates cellattachment and infection.

General procedures for recovery of non-segmented negative-stranded RNAviruses according to the invention can be summarized as follows. Acloned DNA equivalent (which is positive-strand, message sense) of thedesired viral genome is placed between a suitable DNA-dependent RNApolymerase promoter (e.g., a T7, T3 or SP6 RNA polymerase promoter) anda self-cleaving ribozyme sequence (e.g., the hepatitis delta ribozyme)which is inserted into a suitable transcription vector (e.g. apropagatable bacterial plasmid). This transcription vector provides thereadily manipulable DNA template from which the RNA polymerase (e.g., T7RNA polymerase) can faithfully transcribe a single-stranded RNA copy ofthe viral antigenome (or genome) with the precise, or nearly precise, 5′and 3′ termini. The orientation of the viral DNA copy of the genome andthe flanking promoter and ribozyme sequences determine whetherantigenome or genome RNA equivalents are transcribed.

Also required for rescue of new virus progeny according to the inventionare virus-specific trans-acting support proteins needed to encapsidatethe naked, single-stranded viral antigenome or genome RNA transcriptsinto functional nucleocapsid templates. These generally include theviral nucleocapsid (N) protein, the polymerase-associated phosphoprotein(P) and the polymerase (L) protein.

Functional nucleocapsid serves as a template for genome replication,transcription of all viral mRNAs, and accumulation of viral proteins,triggering ensuing events in the viral replication cycle including virusassembly and budding. The mature virus particles contain the viral RNApolymerase necessary for further propagation in susceptible cells.

Certain attenuated viruses selected for rescue require the addition ofsupport proteins, such as G and M for virus assembly and budding. Forexample, the attenuated VSV may be a propagation-defective VSV vectorcomprising a deletion of sequence encoding either all of the G protein(ΔG) or most of the G protein ectodomain (Gstem). Both ΔG and Gstem areunable to spread beyond primary infected cells in vivo. This results ina virus that can propagate only in the presence of transcomplementing Gprotein. Typically, although not necessarily exclusively, rescue ofnon-segmented negative-stranded RNA viruses also requires an RNApolymerase to be expressed in host cells carrying the viral cDNA, todrive transcription of the cDNA-containing transcription vector and ofthe vectors encoding the support proteins. Within the present invention,rescue of attenuated VSV typically involves transfecting host cellswith: a viral cDNA expression vector containing a polynucleotideencoding a genome or antigenome of the attenuated VSV; one or moresupport plasmids encoding N, P, L and G proteins of VSV; and a plasmidencoding a DNA-dependent RNA polymerase, such as T7 RNA polymerase. TheVSV G protein encoded by the support plasmid employed during viralrescue may be encoded by a native VSV G gene. However, the VSV G proteinof a support plasmid used during viral rescue may be encoded by anoptimized VSV G gene. In some embodiments, the cells are alsotransfected with a support plasmid encoding an M protein of VSV. Thetransfected cells are grown in culture, and attenuated VSV is rescuedfrom the culture. The rescued material may then be co-cultured withplaque expansion cells for further viral expansion, as described infurther detail below.

The host cells used for viral rescue are often impaired in their abilityto support further viral expansion. Therefore, the method of producingattenuated VSV in a cell culture typically further includes infectingplaque expansion cells with the rescued, attenuated VSV. In someembodiments of the present invention, cells expressing VSV G proteinencoded by an optimized VSV G gene are infected with the rescuedattenuated VSV; the infected cells are grown; and the attenuated VSV isrecovered from the culture of infected cells.

In some embodiments of viral rescue, the polynucleotide encoding thegenome or antigenome of the attenuated VSV is introduced into the cellin the form of a viral cDNA expression vector that includes thepolynucleotide operatively linked to an expression control sequence todirect synthesis of RNA transcripts from the cDNA expression vector. Insome embodiments, the expression control sequence is a suitableDNA-dependent RNA polymerase promoter (e.g., a 17, T3 or SP6 RNApolymerase promoter). In some embodiments, the support plasmids, as wellas the viral cDNA expression vector used during viral rescue are underthe control of a promoter of the DNA-dependent RNA polymerase. Forexample, in embodiments where the RNA polymerase is T7 RNA polymerase,the support plasmids and the viral cDNA expression vector wouldpreferably be under the control of a T7 promoter. In some otherembodiments, the expression of the DNA-dependent RNA polymerase is underthe control of a cytomegalovirus-derived RNA polymerase Il promoter. Theimmediate-early human cytomegalovirus [hCMV] promoter and enhancer isdescribed, for e.g., in U.S. Pat. No. 5,168,062, incorporated herein byreference.

In some embodiments, the method for recovering attenuated VSV from cDNAinvolves introducing a viral cDNA expression vector encoding a genome orantigenome of the subject virus into a host cell, and coordinatelyintroducing: a polymerase expression vector encoding and directingexpression of an RNA polymerase. Useful RNA polymerases in this contextinclude, but are not limited to, a T7, T3, or SP6 phage polymerase. Thehost cells also express, before, during, or after coordinateintroduction of the viral cDNA expression vector, the polymeraseexpression vector and the N, P, L, M and G support proteins necessaryfor production of mature attenuated VSV particles in the host cell.Typically, the viral cDNA expression vector and polymerase expressionvector will be coordinately transfected into the host cell with one ormore additional expression vector(s) that encode(s) and direct(s)expression of the support proteins. The support proteins may bewild-type or mutant proteins of the virus being rescued, or may beselected from corresponding support protein(s) of a heterologousnon-segmented negative-stranded RNA virus. In alternate embodiments,additional viral proteins may be co-expressed in the host cell, forexample a polymerase elongation factor (such as M2-1 for RSV) or otherviral proteins that may enable or enhance recovery or provide otherdesired results within the subject methods and compositions. In otherembodiments, one or more of the support protein(s) may be expressed inthe host cell by constitutively expressing the protein(s) in the hostcell, or by co-infection of the host cell with a helper virus encodingthe support protein(s).

The viral cDNA vector is introduced into a host cell transientlyexpressing an RNA polymerase and the following VSV support proteins: anN protein, a P protein, an L protein, an M protein and a G protein. Eachof the RNA polymerase and the N, P, L, M and G proteins may be expressedfrom one or more transfected expression vector(s). Often, each of theRNA polymerase and the support proteins will be expressed from separateexpression vectors, commonly from transient expression plasmids.Following a sufficient time and under suitable conditions, an assembledinfectious, attenuated VSV is rescued from the host cells.

To produce infectious, attenuated VSV particles from a cDNA-expressedgenome or antigenome, the genome or antigenome is coexpressed with thoseviral proteins necessary to produce a nucleocapsid capable of RNAreplication, and render progeny nucleocapsids competent for both RNAreplication and transcription. Such viral proteins include the N, P andL proteins. In the instant invention, attenuated VSV vectors with lost Gfunction also require the addition of the G viral protein. Moreover, anM protein may also be added for a productive infection. The G and Mviral proteins can be supplied by coexpression. In some embodiments, theVSV G support plasmid employed during viral rescue contains anon-optimized VSV G gene. However, in other embodiments, as describedbelow, the VSV G support plasmid employed during viral rescue containsan optimized VSV G gene.

In certain embodiments of the invention, complementing sequencesencoding proteins necessary to generate a transcribing, replicatingviral nucleocapsid (i.e., L, P and N), as well as the M and G proteinsare provided by expression plasmids. In other embodiments, such proteinsare provided by one or more helper viruses. Such helper viruses can bewild type or mutant. In certain embodiments, the helper virus can bedistinguished phenotypically from the virus encoded by the recombinantviral cDNA. For example, it may be desirable to provide monoclonalantibodies that react immunologically with the helper virus but not thevirus encoded by the recombinant viral cDNA. Such antibodies can beneutralizing antibodies. In some embodiments, the antibodies can be usedin affinity chromatography to separate the helper virus from therecombinant virus. To aid the procurement of such antibodies, mutationscan be introduced into the viral cDNA to provide antigenic diversityfrom the helper virus, such as in a glycoprotein gene.

A recombinant viral genome or antigenome may be constructed for use inthe present invention by, e.g., assembling cloned cDNA segments,representing in aggregate the complete genome or antigenome, bypolymerase chain reaction or the like (PCR; described in, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methodsand Applications, Innis et al., eds., Academic Press, San Diego, 1990)of reverse-transcribed copies of viral mRNA or genome RNA. For example,a first construct may be generated which comprises cDNAs containing theleft hand end of the antigenome, spanning from an appropriate promoter(e.g., T7, T3, or SP6 RNA polymerase promoter) and assembled in anappropriate expression vector (such as a plasmid, cosmid, phage, or DNAvirus vector). The vector may be modified by mutagenesis and/orinsertion of a synthetic polylinker containing unique restriction sitesdesigned to facilitate assembly. The right hand end of the antigenomeplasmid may contain additional sequences as desired, such as a flankingribozyme and single or tandem T7 transcriptional terminators. Theribozyme can be hammerhead type, which would yield a 3′ end containing asingle nonviral nucleotide, or can be any of the other suitableribozymes such as that of hepatitis delta virus (Perrotta et al., Nature350:434-436, 1991) that would yield a 31 end free of non-viralnucleotides.

Alternative means to construct cDNA encoding the viral genome orantigenome include reverse transcription-PCR using improved PCRconditions (e.g., as described in Cheng et al., Proc. Natl. Acad. Sci.USA 91/5695-5699, 1994, incorporated herein by reference) to reduce thenumber of subunit cDNA components to as few as one or two pieces. Inother embodiments different promoters can be used (e.g., T3 or SPQ).Different DNA vectors (e.g., cosmids) can be used for propagation tobetter accommodate the larger size genome or antigenome.

As noted above, defined mutations can be introduced into an infectiousviral clone by a variety of conventional techniques (e.g., site-directedmutagenesis) into a cDNA copy of the genome or antigenome. The use ofgenomic or antigenomic cDNA subfragments to assemble a complete genomeor antigenome cDNA as described herein has the advantage that eachregion can be manipulated separately, where small cDNA constructsprovide for better ease of manipulation than large cDNA constructs, andthen readily assembled into a complete cDNA.

Certain of the attenuated viruses of the invention will be constructedor modified to limit the growth potential, replication competence, orinfectivity of the recombinant virus. Such attenuated viruses andsubviral particles are useful as vectors and immunogens, but do not posecertain risks that would otherwise attend administration of a fullyinfectious (i.e., having approximately a wild-type level of growthand/or replication competence) virus to a host. By attenuated, it ismeant a virus or subviral particle that is limited in its ability togrow or replicate in a host cell or a mammalian subject, or is otherwisedefective in its ability to infect and/or propagate in or between cells.By way of example, ΔG and G stem are attenuated viruses that arepropagation-defective, but replication competent. Often, attenuatedviruses and subviral particles will be employed as “vectors”, asdescribed in detail herein below.

Thus, various methods and compositions are provided for producingattenuated VSV particles. In more detailed embodiments, the attenuatedvirus will exhibit growth, replication and/or infectivitycharacteristics that are substantially impaired in comparison to growth,replication and/or infectivity of a corresponding wild-type or parentalvirus. In this context, growth, replication, and/or infectivity may beimpaired in vitro and/or in vivo by at least approximately 10-20%,20-50%, 50-75% and up to 95% or greater compared to wild-type orparental growth, replication and/or infectivity levels. In someembodiments, viruses with varying degrees of growth or replicationdefects may be rescued using a combined heat shock/T7-plasmid rescuesystem described in detail below. Exemplary strains include highlyattenuated strains of VSV that incorporate modifications as describedbelow (e.g., a C-terminal G protein truncation, or translocated genes)(see, e.g., Johnson et al., J. Virol. 71:5060-5078, 1997, Schnell etal., Proc. Natl. Acad. Sci. USA 93:11359-11365, 1996; Schnell et al.,Cell 90:849-857, 1997; Roberts et al., J. Virol. 72:4704-4711, 1998; andRose et al., Cell 0.106:539-549, 2001, each incorporated herein byreference).

The VSVs of U.S. Pat. Nos. 7,468,274; 7,419,829; 7,419,674; 7,344,838;7,332,316; 7,329,807; 7,323,337; 7,259,015; 7,244,818; 7,226,786;7,211,247; 7,202,079; 7,198,793; 7,198,784; 7,153,510; 7,070,994;6,969,598; 6,958,226; RE38,824; PP15,957; 6,890,735; 6,887,377;6,867,326; 6,867,036; 6,858,205; 6,835,568; 6,830,892; 6,818,209;6,790,641; 6,787,520; 6,743,620; 6,740,764; 6,740,635; 6,740,320;6,682,907; 6,673,784; 6,673,572; 6,669,936; 6,653,103; 6,607,912;6,558,923; 6,555,107; 6,533,855; 6,531,123; 6,506,604; 6,500,623;6,497,873; 6,489,142; 6,410,316; 6,410,313; 6,365,713; 6,348,312;6,326,487; 6,312,682; 6,303,331; 6,277,633; 6,207,455; 6,200,811;6,190,650; 6,171,862; 6,143,290; 6,133,027; 6,121,434; 6,103,462;6,069,134; 6,054,127; 6,034,073; 5,969,211; 5,935,822; 5,888,727;5,883,081; 5,876,727; 5,858,740; 5,843,723; 5,834,256; 5,817,491;5,792,604; 5,789,229; 5,773,003; 5,763,406; 5,760,184; 5,750,396;5,739,018; 5,698,446; 5,686,279; 5,670,354; 5,540,923; 5,512,421;5,090,194; 4,939,176; 4,738,846; 4,622,292; 4,556,556 and 4,396,628 maybe contemplated by the present invention.

Canine distemper virus (CDV) is a member of the Morbillivirus genus,which also includes measles virus (MV), rinderpest virus (RPV), pestedes petits ruminants virus and morbilliviruses that infect aquaticmammals. CDV infection has been observed in monkey colonies indicatingthat its host range can extend to, but so far, there is no conclusiveevidence linking CDV to human disease in spite of its speculativeassociation to illness of unknown etiology. Lab-adapted CDV has beeninjected into humans without causing symptoms of infection suggestingthat humans are a non-permissive host for CDV, which is consistent withrecent studies showing that mutations facilitating both entry andreplication are needed for CDV to efficiently adapt to human cells.

CDV enters host cells through attachment of H to specific cell receptorsand subsequent F-mediated fusion of viral envelope and cell membrane.Wild-type CDV isolates primarily target signaling lymphocyte activationmolecule (SLAM) and nectin-4 positive cells while vaccine strains of CDVgain broader cell tropisms besides recognizing these two receptors (ref1: The morbillivirus receptor SLAM (CD150). Tatsuo H, Yanagi Y.Microbiol Immunol. 2002; 46(3):135-42. Ref 2: Dog nectin-4 is anepithelial cell receptor for canine distemper virus that facilitatesvirus entry and syncytia formation. Noyce R S, Delpeut S, Richardson CD. Virology. 2013 Feb. 5; 436(1):210-20). Therefore, cell tropisms ofCDV vectors differ depending on usage of wild-type or vaccine CDV Hproteins. In addition, extra specificity determinants can be added to Hprotein ectodomain for specific cancer cell targeting and naturalreceptor interactions deactivated by H mutations, which has beendeveloped in MV-based oncolytic vector research (ref: Paramyxovirusentry and targeted vectors for cancer therapy. Cattaneo R, PLoS Pathog.2010 Jun. 24; 6(6)). Cell retargeting can also be achieved through Fmodifications. Because F function is activated after protease cleavage,paramyxovirus vectors including MV and Sendai virus can be modified toretarget cancer cells through cancer-specific cleavage of F (ref 1:Generation of a recombinant Sendai virus that is selectively activatedand lyses human tumor cells expressing matrix metalloproteinases. KinohH, Inoue M, Washizawa K, Yamamoto T, Fujikawa S, et al. Gene Ther. 2004;11:1137-1145. Ref 2: Oncolytic efficacy and enhanced safety of measlesvirus activated by tumor-secreted matrix metalloproteinases. SpringfeldC, von Messling V, Frenzke M, Ungerechts G, Buchholz C J, Cattaneo R.Cancer Res. 2006; 66:7694-7700). CDV polymerase protein L has genometranscription and replication functions. Modifications in L of vaccineor oncolytic CDV vectors can change viral replication ability, which canserve as a tool to modulate level of CDV attenuation (ref: Developmentof a challenge-protective vaccine concept by modification of the viralRNA-dependent RNA polymerase of canine distemper virus. Silin D,Lyubomska O, Ludlow M, Duprex W P, Rima B K. J Virol. 2007 December;81(24):13649-58).

Recombinant strains of CDV may be developed, for example, as describedby, Miura R, Kooriyama T, Yoneda M, Takenaka A, Doki M, Goto Y, et al.(2015) Efficacy of Recombinant Canine Distemper Virus ExpressingLeishmania Antigen against Leishmania Challenge in Dogs. PLoS Negl TropDis 9(7): e0003914. doi:10.1371/journal.pntd.0003914. Briefly, anantigen of interest may be introduced into a restriction site of afull-length cDNA of a CDV strain RNA genome. CDV rescue may beaccomplished by transfecting HEK293 cells infected with MVA-TV with afull-genome plasmid, together with expression plasmids encoding viralnucleoprotein (N), phosphoprotein (P), and large protein (L) (pKS-N,pKS-P, and pGEM-L, respectively), using FuGENE6 Transfection Reagent(Invitrogen, Carlsbad, Calif., USA). Two days later, the transfectedHEK293 cells were overlain with B95a cells. Syncytia formed by therescued viruses were observed approximately 3 days later. The viruseswere harvested, and their titers determined with the limiting dilutionmethod and expressed as the 50% tissue culture infective dose (TCID₅₀).

The CDVs of U.S. Pat. Nos. 9,526,780; 9,505,812; 9,505,807; 9,327,137;8,709,713; 8,309,531; 7,951,587; 6,664,066; 6,497,882; 6,368,603;6,328,975; 6,309,647; 6,172,979; 5,843,456 and 5,756,102 may becontemplated by the present invention.

The nucleotide sequences and vectors of the invention can be deliveredto cells, for example if aim is to express and the HIV-1 antigens incells in order to produce and isolate the expressed proteins, such asfrom cells grown in culture. For expressing the proteins in cells anysuitable transfection, transformation, or gene delivery methods can beused. Such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used. For example, transfection, transformation, microinjection,infection, electroporation, lipofection, or liposome-mediated deliverycould be used. Expression of the proteins can be carried out in anysuitable type of host cells, such as bacterial cells, yeast, insectcells, and mammalian cells. The proteins of the invention can also beexpressed using including in vitro transcription/translation systems.All of such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used.

In preferred embodiments, the nucleotide sequences, proteins of theinvention are administered in vivo, for example where the aim is toproduce an immunogenic response in a subject. A “subject” in the contextof the present invention may be any animal. For example, in someembodiments it may be desired to express the transgenes of the inventionin a laboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the proteins of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. In preferred embodiments the subject is a human, for examplea human that is infected with, or is at risk of infection with, HIV-1.

For such in vivo applications the nucleotide sequences, proteins of theinvention are preferably administered as a component of an immunogeniccomposition which may comprise the nucleotide sequences and/or antigensof the invention in admixture with a pharmaceutically acceptablecarrier. The immunogenic compositions of the invention are useful tostimulate an immune response against HIV-1 and may be used as one ormore components of a prophylactic or therapeutic vaccine against HIV-1for the prevention, amelioration or treatment of AIDS. The nucleic acidsand vectors of the invention are particularly useful for providinggenetic vaccines, i.e. vaccines for delivering the nucleic acidsencoding the proteins of the invention to a subject, such as a human,such that the proteins are then expressed in the subject to elicit animmune response.

The compositions of the invention may be injectable suspensions,solutions, sprays, lyophilized powders, syrups, elixirs and the like.Any suitable form of composition may be used. To prepare such acomposition, a nucleic acid or vector of the invention, having thedesired degree of purity, is mixed with one or more pharmaceuticallyacceptable carriers and/or excipients. The carriers and excipients mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to, water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, or combinations thereof,buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN′, PLURONICS' or polyethylene glycol (PEG).

An immunogenic or immunological composition can also be formulated inthe form of an oil-in-water emulsion. The oil-in-water emulsion can bebased, for example, on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane, squalene, EICOSANE™ ortetratetracontane; oil resulting from the oligomerization of alkene(s),e.g., isobutene or decene; esters of acids or of alcohols containing alinear alkyl group, such as plant oils, ethyl oleate, propylene glycoldi(caprylate/caprate), glyceryl tri(caprylate/caprate) or propyleneglycol dioleate; esters of branched fatty acids or alcohols, e.g.,isostearic acid esters. The oil advantageously is used in combinationwith emulsifiers to form the emulsion. The emulsifiers can be nonionicsurfactants, such as esters of sorbitan, mannide (e.g., anhydromannitololeate), glycerol, polyglycerol, propylene glycol, and oleic,isostearic, ricinoleic, or hydroxystearic acid, which are optionallyethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, suchas the Pluronic® products, e.g., L121. The adjuvant can be a mixture ofemulsifier(s), micelle-forming agent, and oil such as that which iscommercially available under the name Provax® (IDEC Pharmaceuticals, SanDiego, Calif.).

The immunogenic compositions of the invention can contain additionalsubstances, such as wetting or emulsifying agents, buffering agents, oradjuvants to enhance the effectiveness of the vaccines (Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.)1980).

Adjuvants may also be included. Adjuvants include, but are not limitedto, mineral salts (e.g., AlK(SO₄)₂, AlNa(SO₄)₂, AlNH(SO₄)₂, silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, or carbon), polynucleotides with orwithout immune stimulating complexes (ISCOMs) (e.g., CpGoligonucleotides, such as those described in Chuang, T. H. et al, (2002)J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al (2002) Eur. J.Immunol. 32(7): 1958-68; poly IC or poly AU acids, polyarginine with orwithout CpG (also known in the art as IC31; see Schellack, C. et al(2003) Proceedings of the 34th Annual Meeting of the German Society ofImmunology; Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508),JuvaVax™ (U.S. Pat. No. 6,693,086), certain natural substances (e.g.,wax D from Mycobacterium tuberculosis, substances found inCornyebacterium parvum, Bordetella pertussis, or members of the genusBrucella), flagellin (Toll-like receptor 5 ligand; see McSorley, S. J.et al (2002) J. Immunol. 169(7): 3914-9), saponins such as QS21, QS17,and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495),monophosphoryl lipid A, in particular, 3-de-O-acylated monophosphoryllipid A (3D-MPL), imiquimod (also known in the art as IQM andcommercially available as Aldara®; U.S. Pat. Nos. 4,689,338; 5,238,944;Zuber, A. K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitorCMPD167 (see Veazey, R. S. et al (2003) J. Exp. Med. 198: 1551-1562).

Aluminum hydroxide or phosphate (alum) are commonly used at 0.05 to 0.1%solution in phosphate buffered saline. Other adjuvants that can be used,especially with DNA vaccines, are cholera toxin, especiallyCTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J. Immunol. 167(6):3398-405), polyphosphazenes (Allcock, H. R. (1998) App. OrganometallicChem. 12(10-11): 659-666; Payne, L. G. et al (1995) Pharm. Biotechnol.6: 473-93), cytokines such as, but not limited to, IL-2, IL-4, GM-CSF,IL-12, IL-15 IGF-1, IFN-α, IFN-β, and IFN-γ (Boyer et al., (2002) J.Liposome Res. 121:137-142; WO01/095919), immunoregulatory proteins suchas CD4OL (ADX40; see, for example, WO03/063899), and the CD1a ligand ofnatural killer cells (also known as CRONY or α-galactosyl ceramide; seeGreen, T. D. et al, (2003) J. Virol. 77(3): 2046-2055),immunostimulatory fusion proteins such as IL-2 fused to the Fc fragmentof immunoglobulins (Barouch et al., Science 290:486-492, 2000) andco-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can beadministered either as proteins or in the form of DNA, on the sameexpression vectors as those encoding the antigens of the invention or onseparate expression vectors.

In an advantageous embodiment, the adjuvants may be lecithin is combinedwith an acrylic polymer (Adjuplex-LAP), lecithin coated oil droplets inan oil-in-water emulsion (Adjuplex-LE) or lecithin and acrylic polymerin an oil-in-water emulsion (Adjuplex-LAO) (Advanced BioAdjuvants(ABA)).

The immunogenic compositions can be designed to introduce the nucleicacids or expression vectors to a desired site of action and release itat an appropriate and controllable rate. Methods of preparingcontrolled-release formulations are known in the art. For example,controlled release preparations can be produced by the use of polymersto complex or absorb the immunogen and/or immunogenic composition. Acontrolled-release formulation can be prepared using appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) known to provide thedesired controlled release characteristics or release profile. Anotherpossible method to control the duration of action by acontrolled-release preparation is to incorporate the active ingredientsinto particles of a polymeric material such as, for example, polyesters,polyamino acids, hydrogels, polylactic acid, polyglycolic acid,copolymers of these acids, or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these active ingredients intopolymeric particles, it is possible to entrap these materials intomicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacrylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed in NewTrends and Developments in Vaccines, Voller et al. (eds.), UniversityPark Press, Baltimore, Md., 1978 and Remington's PharmaceuticalSciences, 16th edition.

Suitable dosages of the nucleic acids and expression vectors of theinvention (collectively, the immunogens) in the immunogenic compositionof the invention can be readily determined by those of skill in the art.For example, the dosage of the immunogens can vary depending on theroute of administration and the size of the subject. Suitable doses canbe determined by those of skill in the art, for example by measuring theimmune response of a subject, such as a laboratory animal, usingconventional immunological techniques, and adjusting the dosages asappropriate. Such techniques for measuring the immune response of thesubject include but are not limited to, chromium release assays,tetramer binding assays, IFN-γ ELISPOT assays, IL-2 ELISPOT assays,intracellular cytokine assays, and other immunological detection assays,e.g., as detailed in the text “Antibodies: A Laboratory Manual” by EdHarlow and David Lane.

When provided prophylactically, the immunogenic compositions of theinvention are ideally administered to a subject in advance of HIVinfection, or evidence of HIV infection, or in advance of any symptomdue to AIDS, especially in high-risk subjects. The prophylacticadministration of the immunogenic compositions can serve to provideprotective immunity of a subject against HIV-1 infection or to preventor attenuate the progression of AIDS in a subject already infected withHIV-1. When provided therapeutically, the immunogenic compositions canserve to ameliorate and treat AIDS symptoms and are advantageously usedas soon after infection as possible, preferably before appearance of anysymptoms of AIDS but may also be used at (or after) the onset of thedisease symptoms.

The immunogenic compositions can be administered using any suitabledelivery method including, but not limited to, intramuscular,intravenous, intradermal, mucosal, and topical delivery. Such techniquesare well known to those of skill in the art. More specific examples ofdelivery methods are intramuscular injection, intradermal injection, andsubcutaneous injection. However, delivery need not be limited toinjection methods. Further, delivery of DNA to animal tissue has beenachieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod.Dev. 38:268-274; and WO 96/20013), direct injection of naked DNA intoanimal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960;Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994)Virology 199: 132-140; Webster et al., (1994) Vaccine 12: 1495-1498;Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et al., (1993)Hum. Mol. Gen. 2: 1847-1851), or intradermal injection of DNA using“gene gun” technology (Johnston et al., (1994) Meth. Cell Biol.43:353-365). Alternatively, delivery routes can be oral, intranasal orby any other suitable route. Delivery also be accomplished via a mucosalsurface such as the anal, vaginal or oral mucosa.

Immunization schedules (or regimens) are well known for animals(including humans) and can be readily determined for the particularsubject and immunogenic composition. Hence, the immunogens can beadministered one or more times to the subject. Preferably, there is aset time interval between separate administrations of the immunogeniccomposition. While this interval varies for every subject, typically itranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.For humans, the interval is typically from 2 to 6 weeks. Theimmunization regimes typically have from 1 to 6 administrations of theimmunogenic composition, but may have as few as one or two or four. Themethods of inducing an immune response can also include administrationof an adjuvant with the immunogens. In some instances, annual, biannualor other long interval (5-10 years) booster immunization can supplementthe initial immunization protocol.

The present methods also include a variety of prime-boost regimens, forexample DNA prime-Adenovirus boost regimens. In these methods, one ormore priming immunizations are followed by one or more boostingimmunizations. The actual immunogenic composition can be the same ordifferent for each immunization and the type of immunogenic composition(e.g., containing protein or expression vector), the route, andformulation of the immunogens can also be varied. For example, if anexpression vector is used for the priming and boosting steps, it caneither be of the same or different type (e.g., DNA or bacterial or viralexpression vector). One useful prime-boost regimen provides for twopriming immunizations, four weeks apart, followed by two boostingimmunizations at 4 and 8 weeks after the last priming immunization. Itshould also be readily apparent to one of skill in the art that thereare several permutations and combinations that are encompassed using theDNA, bacterial and viral expression vectors of the invention to providepriming and boosting regimens.

The prime-boost regimen can also include VSV vectors that derive their Gprotein protein from different serotype vesicular stomatitis viruses(Rose N F, Roberts A, Buonocore L, Rose J K. Glycoprotein exchangevectors based on vesicular stomatitis virus allow effective boosting andgeneration of neutralizing antibodies to a primary isolate of humanimmunodeficiency virus type 1. J Virol. 2000 December; 74(23):10903-10).The VSV vectors used in these examples contain a G protein derived fromthe Indiana serotype of VSV. Vectors can also be constructed to expressepitopes in the context of G molecules derived from other VSV serotypes(i.e. vesicular stomatitis New Jersey virus or vesicular stomatitisAlagoas virus) or other vesiculoviruses (i.e. Chandipura virus, Cocalvirus, Isfahan virus). Thus an epitope like the HIV MPER can bedelivered in a prime in the context of a G molecule that is from theIndiana serotype and the immune system can be boosted with a vector thatexpresses epitopes in the context of second serotype like New Jersey.This circumvents anti-G immunity elicited by the prime, and helps focusthe boost response agains the foreign epitope.

A specific embodiment of the invention provides methods of inducing animmune response against HIV in a subject by administering an immunogeniccomposition of the invention, preferably which may comprise anadenovirus vector containing DNA encoding one or more of the epitopes ofthe invention, one or more times to a subject wherein the epitopes areexpressed at a level sufficient to induce a specific immune response inthe subject. Such immunizations can be repeated multiple times at timeintervals of at least 2, 4 or 6 weeks (or more) in accordance with adesired immunization regime.

The immunogenic compositions of the invention can be administered alone,or can be co-administered, or sequentially administered, with other HIVimmunogens and/or HIV immunogenic compositions, e.g., with “other”immunological, antigenic or vaccine or therapeutic compositions therebyproviding multivalent or “cocktail” or combination compositions of theinvention and methods of employing them. Again, the ingredients andmanner (sequential or co-administration) of administration, as well asdosages can be determined taking into consideration such factors as theage, sex, weight, species and condition of the particular subject, andthe route of administration.

When used in combination, the other HIV immunogens can be administeredat the same time or at different times as part of an overallimmunization regime, e.g., as part of a prime-boost regimen or otherimmunization protocol. In an advantageous embodiment, the other HIVimmunogen is env, preferably the HIV env trimer.

Many other HIV immunogens are known in the art, one such preferredimmunogen is HIVA (described in WO 01/47955), which can be administeredas a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g.,MVA.HIVA). Another such HIV immunogen is RENTA (described inPCT/US2004/037699), which can also be administered as a protein, on aplasmid (e.g., pTHr.RENTA) or in a viral vector (e.g., MVA.RENTA).

For example, one method of inducing an immune response against HIV in ahuman subject may comprise administering at least one priming dose of anHIV immunogen and at least one boosting dose of an HIV immunogen,wherein the immunogen in each dose can be the same or different,provided that at least one of the immunogens is an epitope of thepresent invention, a nucleic acid encoding an epitope of the inventionor an expression vector, preferably a VSV vector, encoding an epitope ofthe invention, and wherein the immunogens are administered in an amountor expressed at a level sufficient to induce an HIV-specific immuneresponse in the subject. The HIV-specific immune response can include anHIV-specific T-cell immune response or an HIV-specific B-cell immuneresponse. Such immunizations can be done at intervals, preferably of atleast 0-29 or more weeks.

Preclinical efficacy in the SHIV challenge model was observed followingmucosal vaccination with a total dose of 2×10⁸ pfu per ml. The vaccinedose may be split between two sites, such as mucosal surfaces in thenasal and oral cavities, where each received 1×10⁸ pfu applied in abuffered solution. Dosages ranging from about 1×10⁴ to 1×10⁹ pfu per mlare also contemplated. Single doses are also contemplated.Alternatively, a vaccination schedule from about 0 to 40 weeks iscontemplated. A vaccination schedule may be at 0, 4 and 29 weeks.

Preclinical efficacy in the SHIV challenge model was observed with avaccination schedule of 0, 4, and 29 weeks. Other vaccination schedulesare also contemplated.

SEQ ID NO: 1: 1aaattaatac gactcactat agggagacca caacggtttc cctctagcgt tgtcttcgtc 61tgatgagtcc gtgaggacga aactatagga aaggaattcc tatagtcACG AAGACAAACA 121AACCATTATT ATCATTAAAA GGCTCAGGAG AAACTTTAAC AGTAATCAAA ATGTCTGTTA 181CAGTCAAGAG AATCATTGAC AACACAGTCA TAGTTCCAAA ACTTCCTGCA AATGAGGATC 241CAGTGGAATA CCCGGCAGAT TACTTCAGAA AATCAAAGGA GATTCCTCTT TACATCAATA 301CTACAAAAAG TTTGTCAGAT CTAAGAGGAT ATGTCTACCA AGGCCTCAAA TCCGGAAATG 361TATCAATCAT ACATGTCAAC AGCTACTTGT ATGGAGCATT GAAGGACATC CGGGGTAAGT 421TGGATAAAGA TTGGTCAAGT TTCGGAATAA ACATCGGGAA GGCAGGGGAT ACAATCGGAA 481TATTTGACCT TGTATCCTTG AAAGCCCTGG ACGGTGTACT TCCAGATGGA GTATCGGATG 541CTTCCAGAAC CAGCGCAGAT GACAAATGGT TGCCTTTGTA TCTACTTGGC TTATACAGAG 601TGGGCAGAAC ACAAATGCCT GAATACAGAA AAAGGCTCAT GGATGGGCTG ACAAATCAAT 661GCAAAATGAT CAATGAACAG TTTGAACCTC TTGTGCCAGA AGGTCGTGAC ATTTTTGATG 721TGTGGGGAAA TGACAGTAAT TACACAAAAA TTGTCGCTGC AGTGGACATG TTCTTCCACA 781TGTTCAAAAA ACATGAATGT GCCTCGTTCA GATACGGAAC TATTGTTTCC AGATTCAAAG 841ATTGTGCTGC ATTGGCAACA TTTGGACACC TCTGCAAAAT AACCGGAATG TCTACAGAAG 901ATGTGACGAC CTGGATCTTG AACCGAGAAG TTGCAGATGA GATGGTCCAA ATGATGCTTC 961CAGGCCAAGA AATTGACAAG GCTGATTCAT ACATGCCTTA TTTGATCGAC TTTGGATTGT 1021CTTCTAAGTC TCCATATTCT TCCGTCAAAA ACCCTGCCTT CCACTTCTGG GGGCAATTGA 1081CAGCTCTTCT GCTCAGATCC ACCAGAGCAA GGAATGCCCG ACAGCCTGAT GACATTGAGT 1141ATACATCTCT TACTACAGCA GGTTTGTTGT ACGCTTATGC AGTAGGATCC TCTGCTGACT 1201TGGCACAACA GTTTTGTGTT GGAGATAGCA AATACACTCC AGATGATAGT ACCGGAGGAT 1261TGACGACTAA TGCACCGCCA CAAGGCAGAG ATGTGGTCGA ATGGCTCGGA TGGTTTGAAG 1321ATCAAAACAG AAAACCGACT CCTGATATGA TGCAGTATGC GAAACGAGCA GTCATGTCAC 1381TGCAAGGCCT AAGAGAGAAG ACAATTGGCA AGTATGCTAA GTCAGAGTTT GACAAATGAC 1441CCTATAATTC TCAGATCACC TATTATATAT TATGCTAGCT ATGAAAAAAA CTAACAGATA 1501TCATGGATAA TCTCACAAAA GTTCGTGAGT ATCTCAAGTC CTATTCTCGT CTAGATCAGG 1561CGGTAGGAGA GATAGATGAG ATCGAAGCAC AACGAGCTGA AAAGTCCAAT TATGAGTTGT 1621TCCAAGAGGA CGGAGTGGAA GAGCATACTA GGCCCTCTTA TTTTCAGGCA GCAGATGATT 1681CTGACACAGA ATCTGAACCA GAAATTGAAG ACAATCAAGG CTTGTATGTA CCAGATCCGG 1741AAGCTGAGCA AGTTGAAGGC TTTATACAGG GGCCTTTAGA TGACTATGCA GATGAGGACG 1801TGGATGTTGT ATTCACTTCG GACTGGAAAC AGCCTGAGCT TGAATCCGAC GAGCATGGAA 1861AGACCTTACG GTTGACATTG CCAGAGGGTT TAAGTGGAGA GCAGAAATCC CAGTGGCTTT 1921TGACGATTAA AGCAGTCGTT CAAAGTGCCA AACACTGGAA TCTGGCAGAG TGCACATTTG 1981AAGCATCGGG AGAAGGGGTC ATCATAAAAA AGCGCCAGAT AACTCCGGAT GTATATAAGG 2041TCACTCCAGT GATGAACACA CATCCGTCCC AATCAGAAGC CGTATCAGAT GTTTGGTCTC 2101TCTCAAAGAC ATCCATGACT TTCCAACCCA AGAAAGCAAG TCTTCAGCCT CTCACCATAT 2161CCTTGGATGA ATTGTTCTCA TCTAGAGGAG AATTCATCTC TGTCGGAGGT AACGGACGAA 2221TGTCTCATAA AGAGGCCATC CTGCTCGGTC TGAGGTACAA AAAGTTGTAC AATCAGGCGA 2281GAGTCAAATA TTCTCTGTAG ACTAGTATGA AAAAAAGTAA CAGATATCAC AATCTAAGTG 2341TTATCCCAAT CCATTCATCA TGAGTTCCTT AAAGAAGATT CTCGGTCTGA AGGGGAAAGG 2401TAAGAAATCT AAGAAATTAG GGATCGCACC ACCCCCTTAT GAAGAGGACA CTAACATGGA 2461GTATGCTCCG AGCGCTCCAA TTGACAAATC CTATTTTGGA GTTGACGAGA TGGACACTCA 2521TGATCCGAAT CAATTAAGAT ATGAGAAATT CTTCTTTACA GTGAAAATGA CGGTTAGATC 2581TAATCGTCCG TTCAGAACAT ACTCAGATGT GGCAGCCGCT GTATCCCATT GGGATCACAT 2641GTACATCGGA ATGGCAGGGA AACGTCCCTT CTACAAGATC TTGGCTTTTT TGGGTTCTTC 2701TAATCTAAAG GCCACTCCAG CGGTATTGGC AGATCAAGGT CAACCAGAGT ATCATGCTCA 2761CTGTGAAGGC AGGGCTTATT TGCCACACAG AATGGGGAAG ACCCCTCCCA TGCTCAATGT 2821ACCAGAGCAC TTCAGAAGAC CATTCAATAT AGGTCTTTAC AAGGGAACGA TTGAGCTCAC 2881AATGACCATC TACGATGATG AGTCACTGGA AGCAGCTCCT ATGATCTGGG ATCATTTCAA 2941TTCTTCCAAA TTTTCTGATT TCAGAGAGAA GGCCTTAATG TTTGGCCTGA TTGTCGAGAA 3001AAAGGCATCT GGAGCTTGGG TCCTGGATTC TGTCAGCCAC TTCAAATGAG CTAGTCTAGC 3061TTCCAGCTTC TGAACAATCC CCGGTTTACT CAGTCTCTCC TAATTCCAGC CTTTCGAACA 3121ACTAATATCC TGTCTTCTCT ATCCCTATGA AAAAAACTAA CAGAGATCGA TCTGTTTCCT 3181TGACACCAGG AGCCACCATG AAGTGCCTTT TGTACTTAGC TTTTTTATTC ATCGGGGTGA 3241ATTGCAAGGC TAGCGCAGAG AATTTGTGGG TAACAGTCTA CTATGGAGTC CCTGTATGGA 3301AGGATGCAGA GACAACATTG TTCTGTGCTA GTGACGCAAA GGCTTACGAG ACGGAGAAGC 3361ACAATGTGTG GGCAACTCAC GCATGTGTCC CAACCGATCC AAATCCTCAA GAGATTCATC 3421TAGAGAATGT GACTGAAGAA TTCAATATGT GGAAGAATAA TATGGTAGAG CAAATGCATA 3481CAGATATCAT TAGTTTATGG GACCAGTCAC TTAAACCCTG CGTTAAATTG ACGCCTCTAT 3541GTGTGACACT TCAATGTACT AATGTTACAA ACAACATAAC AGATGATATG AGAGGAGAAC 3601TGAAGAACTG TAGTTTCAAC ATGACGACAG AGTTGCGTGA CAAGAAACAG AAAGTGTATT 3661CACTATTCTA TCGGTTGGAT GTAGTACAGA TAAATGAGAA TCAAGGAAAC AGGTCCAACA 3721ACTCTAACAA AGAGTACAGA CTTATTAATT GCAATACCAG TGCTATCACG CAAGCCTGCC 3781CAAAGGTTTC ATTTGAACCA ATACCTATTC ATTATTGTGC ACCTGCTGGA TTCGCCATCC 3841TCAAATGTAA AGACAAGAAG TTCAATGGAA CAGGACCCTG CCCATCAGTT TCAACCGTTC 3901AGTGCACCCA CGGAATCAAG CCTGTAGTTA GTACTCAATT ATTGTTAAAT GGGAGCTTAG 3961CTGAAGAAGA AGTTATGATT AGATCAGAGA ATATTACCAA TAATGCGAAG AACATCTTGG 4021TTCAATTCAA TACTCCAGTC CAGATCAATT GCACAAGGCC TAATAATAAT ACCAGAAAGA 4081GTATAAGAAT TGGGCCAGGA CAGGCATTCT ATGCAACAGG AGATATAATC GGAGACATTC 4141GACAAGCGCA CTGCACTGTT TCTAAGGCCA CTTGGAATGA AACATTGGGT AAAGTTGTAA 4201AGCAACTTCG GAAGCATTTC GGAAATAACA CAATTATTAG ATTTGCGAAC TCATCTGGAG 4261GGGATCTGGA AGTGACAACA CACTCTTTCA ATTGCGGTGG CGAGTTCTTC TATTGTAATA 4321CAAGTGGATT ATTTAACTCT ACTTGGATTT CAAATACCTC AGTCCAAGGA TCTAATTCAA 4381CAGGGTCTAA CGATTCTATA ACATTACCTT GCCGTATAAA GCAAATTATT AATATGTGGC 4441AAAGAATCGG GCAAGCGATG TATGCTCCAC CTATTCAAGG CGTGATTCGT TGCGTTTCAA 4501ACATAACAGG GTTGATCCTG ACCAGGGATG GAGGCTCTAC CAATTCCACC ACCGAGACCT 4561TCCGTCCCGG TGGCGGAGAT ATGCGGGATA ACTGGAGATC AGAGCTCTAT AAGTATAAGG 4621TTGTGAAGAT TGAACCTCTT GGAGTTGCCC CTACAAGAGC AAAGAGAAGG GTGGTTGGCC 4681GAGAGAAGAG AGCAGTTGGC ATCGGTGCTG TCTTTCTCGG ATTTCTTGGA GCAGCTGGAT 4741CCACTATGGG AGCAGCATCA ATGACACTAA CAGTGCAGGC TAGAAATTTG CTTAGCGGAA 4801TCGTTCAGCA GCAGAGCAAT TTACTAAGAG CAATTGAAGC ACAGCAACAT CTCTTAAAGT 4861TGACGGTGTG GGGCATTAAA CAACTACAAG CGAGAGTGCT TGCCGTCGAA AGATATTTGC 4921GAGACCAACA GCTATTGGGT ATTTGGGGTT GTTCTGGGAA ATTAATTTGC ACAACAAATG 4981TTCCATGGAA CTCCTCCTGG AGTAATAGGA ATTTAAGTGA GATATGGGAC AACATGACAT 5041GGTTGCAGTG GGACAAGGAA ATCTCAAATT ATACACAGAT AATCTATGGA TTATTAGAAG 5101AGTCTCAGAA TCAGCAAGAG AAGAATGAAC AGGATTTGCT TGCATTGGAT AAGTGGGCTT 5161CTCTATGGAA CTGGTTCGAT ATTAGTAATT GGCTCTGGTA TATTAAGAGC TCTATTGCCT 5221CTTTTTTCTT TATCATAGGG TTAATCATTG GACTATTCTT GGTTCTCCGA GTTGGTATTT 5281ATCTTTGCAT TAAATTAAAG CACACCAAGA AAAGACAGAT TTATACAGAC ATAGAGATGA 5341ACCGACTTGG AAAGTAAAGC TCAAATCCTG CACAACAGAT TCTTCATGTT TGAACCAAAT 5401CAACTTGTGA TATCATGCTC AAAGAGGCCT TAATTAAATT TTAATTTTTA ATTTTTATGA 5461AAAAAACTAA CAGCAATCAT GGAAGTCCAC GATTTTGAGA CCGACGAGTT CAATGATTTC 5521AATGAAGATG ACTATGCCAC AAGAGAATTC CTGAATCCCG ATGAGCGCAT GACGTACTTG 5581AATCATGCTG ATTACAATTT GAATTCTCCT CTAATTAGTG ATGATATTGA CAATTTGATC 5641AGGAAATTCA ATTCTCTTCC GATTCCCTCG ATGTGGGATA GTAAGAACTG GGATGGAGTT 5701CTTGAGATGT TAACATCATG TCAAGCCAAT CCCATCTCAA CATCTCAGAT GCATAAATGG 5761ATGGGAAGTT GGTTAATGTC TGATAATCAT GATGCCAGTC AAGGGTATAG TTTTTTACAT 5821GAAGTGGACA AAGAGGCAGA AATAACATTT GACGTGGTGG AGACCTTCAT CCGCGGCTGG 5881GGCAACAAAC CAATTGAATA CATCAAAAAG GAAAGATGGA CTGACTCATT CAAAATTCTC 5941GCTTATTTGT GTCAAAAGTT TTTGGACTTA CACAAGTTGA CATTAATCTT AAATGCTGTC 6001TCTGAGGTGG AATTGCTCAA CTTGGCGAGG ACTTTCAAAG GCAAAGTCAG AAGAAGTTCT 6061CATGGAACGA ACATATGCAG GCTTAGGGTT CCCAGCTTGG GTCCTACTTT TATTTCAGAA 6121GGATGGGCTT ACTTCAAGAA ACTTGATATT CTAATGGACC GAAACTTTCT GTTAATGGTC 6181AAAGATGTGA TTATAGGGAG GATGCAAACG GTGCTATCCA TGGTATGTAG AATAGACAAC 6241CTGTTCTCAG AGCAAGACAT CTTCTCCCTT CTAAATATCT ACAGAATTGG AGATAAAATT 6301GTGGAGAGGC AGGGAAATTT TTCTTATGAC TTGATTAAAA TGGTGGAACC GATATGCAAC 6361TTGAAGCTGA TGAAATTAGC AAGAGAATCA AGGCCTTTAG TCCCACAATT CCCTCATTTT 6421GAAAATCATA TCAAGACTTC TGTTGATGAA GGGGCAAAAA TTGACCGAGG TATAAGATTC 6481CTCCATGATC AGATAATGAG TGTGAAAACA GTGGATCTCA CACTGGTGAT TTATGGATCG 6541TTCAGACATT GGGGTCATCC TTTTATAGAT TATTACGCTG GACTAGAAAA ATTACATTCC 6601CAAGTAACCA TGAAGAAAGA TATTGATGTG TCATATGCAA AAGCACTTGC AAGTGATTTA 6661GCTCGGATTG TTCTATTTCA ACAGTTCAAT GATCATAAAA AGTGGTTCGT GAATGGAGAC 6721TTGCTCCCTC ATGATCATCC CTTTAAAAGT CATGTTAAAG AAAATACATG GCCTACAGCT 6781GCTCAAGTTC AAGATTTTGG AGATAAATGG CATGAACTTC CGCTGATTAA ATGTTTTGAA 6841ATACCCGACT TACTAGACCC ATCGATAATA TACTCTGACA AAAGTCATTC AATGAATAGG 6901TCAGAGGTGT TGAAACATGT CCGAATGAAT CCGAACACTC CTATCCCTAG TAAAAAGGTG 6961TTGCAGACTA TGTTGGACAC AAAGGCTACC AATTGGAAAG AATTTCTTAA AGAGATTGAT 7021GAGAAGGGCT TAGATGATGA TGATCTAATT ATTGGTCTTA AAGGAAAGGA GAGGGAACTG 7081AAGTTGGCAG GTAGATTTTT CTCCCTAATG TCTTGGAAAT TGCGAGAATA CTTTGTAATT 7141ACCGAATATT TGATAAAGAC TCATTTCGTC CCTATGTTTA AAGGCCTGAC AATGGCGGAC 7201GATCTAACTG CAGTCATTAA AAAGATGTTA GATTCCTCAT CCGGCCAAGG ATTGAAGTCA 7261TATGAGGCAA TTTGCATAGC CAATCACATT GATTACGAAA AATGGAATAA CCACCAAAGG 7321AAGTTATCAA ACGGCCCAGT GTTCCGAGTT ATGGGCCAGT TCTTAGGTTA TCCATCCTTA 7381ATCGAGAGAA CTCATGAATT TTTTGAGAAA AGTCTTATAT ACTACAATGG AAGACCAGAC 7441TTGATGCGTG TTCACAACAA CACACTGATC AATTCAACCT CCCAACGAGT TTGTTGGCAA 7501GGACAAGAGG GTGGACTGGA AGGTCTACGG CAAAAAGGAT GGAGTATCCT CAATCTACTG 7561GTTATTCAAA GAGAGGCTAA AATCAGAAAC ACTGCTGTCA AAGTCTTGGC ACAAGGTGAT 7621AATCAAGTTA TTTGCACACA GTATAAAACG AAGAAATCGA GAAACGTTGT AGAATTACAG 7681GGTGCTCTCA ATCAAATGGT TTCTAATAAT GAGAAAATTA TGACTGCAAT CAAAATAGGG 7741ACAGGGAAGT TAGGACTTTT GATAAATGAC GATGAGACTA TGCAATCTGC AGATTACTTG 7801AATTATGGAA AAATACCGAT TTTCCGTGGA GTGATTAGAG GGTTAGAGAC CAAGAGATGG 7861TCACGAGTGA CTTGTGTCAC CAATGACCAA ATACCCACTT GTGCTAATAT AATGAGCTCA 7921GTTTCCACAA ATGCTCTCAC CGTAGCTCAT TTTGCTGAGA ACCCAATCAA TGCCATGATA 7981CAGTACAATT ATTTTGGGAC ATTTGCTAGA CTCTTGTTGA TGATGCATGA TCCTGCTCTT 8041CGTCAATCAT TGTATGAAGT TCAAGATAAG ATACCGGGCT TGCACAGTTC TACTTTCAAA 8101TACGCCATGT TGTATTTGGA CCCTTCCATT GGAGGAGTGT CGGGCATGTC TTTGTCCAGG 8161TTTTTGATTA GAGCCTTCCC AGATCCCGTA ACAGAAAGTC TCTCATTCTG GAGATTCATC 8221CATGTACATG CTCGAAGTGA GCATCTGAAG GAGATGAGTG CAGTATTTGG AAACCCCGAG 8281ATAGCCAAGT TCCGAATAAC TCACATAGAC AAGCTAGTAG AAGATCCAAC CTCTCTGAAC 8341ATCGCTATGG GAATGAGTCC AGCGAACTTG TTAAAGACTG AGGTTAAAAA ATGCTTAATC 8401GAATCAAGAC AAACCATCAG GAACCAGGTG ATTAAGGATG CAACCATATA TTTGTATCAT 8461GAAGAGGATC GGCTCAGAAG TTTCTTATGG TCAATAAATC CTCTGTTCCC TAGATTTTTA 8521AGTGAATTCA AATCAGGCAC TTTTTTGGGA GTCGCAGACG GGCTCATCAG TCTATTTCAA 8581AATTCTCGTA CTATTCGGAA CTCCTTTAAG AAAAAGTATC ATAGGGAATT GGATGATTTG 8641ATTGTGAGGA GTGAGGTATC CTCTTTGACA CATTTAGGGA AACTTCATTT GAGAAGGGGA 8701TCATGTAAAA TGTGGACATG TTCAGCTACT CATGCTGACA CATTAAGATA CAAATCCTGG 8761GGCCGTACAG TTATTGGGAC AACTGTACCC CATCCATTAG AAATGTTGGG TCCACAACAT 8821CGAAAAGAGA CTCCTTGTGC ACCATGTAAC ACATCAGGGT TCAATTATGT TTCTGTGCAT 8881TGTCCAGACG GGATCCATGA CGTCTTTAGT TCACGGGGAC CATTGCCTGC TTATCTAGGG 8941TCTAAAACAT CTGAATCTAC ATCTATTTTG CAGCCTTGGG AAAGGGAAAG CAAAGTCCCA 9001CTGATTAAAA GAGCTACACG TCTTAGAGAT GCTATCTCTT GGTTTGTTGA ACCCGACTCT 9061AAACTAGCAA TGACTATACT TTCTAACATC CACTCTTTAA CAGGCGAAGA ATGGACCAAA 9121AGGCAGCATG GGTTCAAAAG AACAGGGTCT GCCCTTCATA GGTTTTCGAC ATCTCGGATG 9181AGCCATGGTG GGTTCGCATC TCAGAGCACT GCAGCATTGA CCAGGTTGAT GGCAACTACA 9241GACACCATGA GGGATCTGGG AGATCAGAAT TTCGACTTTT TATTCCAAGC AACGTTGCTC 9301TATGCTCAAA TTACCACCAC TGTTGCAAGA GACGGATGGA TCACCAGTTG TACAGATCAT 9361TATCATATTG CCTGTAAGTC CTGTTTGAGA CCCATAGAAG AGATCACCCT GGACTCAAGT 9421ATGGACTACA CGCCCCCAGA TGTATCCCAT GTGCTGAAGA CATGGAGGAA TGGGGAAGGT 9481TCGTGGGGAC AAGAGATAAA ACAGATCTAT CCTTTAGAAG GGAATTGGAA GAATTTAGCA 9541CCTGCTGAGC AATCCTATCA AGTCGGCAGA TGTATAGGTT TTCTATATGG AGACTTGGCG 9601TATAGAAAAT CTACTCATGC CGAGGACAGT TCTCTATTTC CTCTATCTAT ACAAGGTCGT 9661ATTAGAGGTC GAGGTTTCTT AAAAGGGTTG CTAGACGGAT TAATGAGAGC AAGTTGCTGC 9721CAAGTAATAC ACCGGAGAAG TCTGGCTCAT TTGAAGAGGC CGGCCAACGC AGTGTACGGA 9781GGTTTGATTT ACTTGATTGA TAAATTGAGT GTATCACCTC CATTCCTTTC TCTTACTAGA 9841TCAGGACCTA TTAGAGACGA ATTAGAAACG ATTCCCCACA AGATCCCAAC CTCCTATCCG 9901ACAAGCAACC GTGATATGGG GGTGATTGTC AGAAATTACT TCAAATACCA ATGCCGTCTA 9961ATTGAAAAGG GAAAATACAG ATCACATTAT TCACAATTAT GGTTATTCTC AGATGTCTTA 10021TCCATAGACT TCATTGGACC ATTCTCTATT TCCACCACCC TCTTGCAAAT CCTATACAAG 10081CCATTTTTAT CTGGGAAAGA TAAGAATGAG TTGAGAGAGC TGGCAAATCT TTCTTCATTG 10141CTAAGATCAG GAGAGGGGTG GGAAGACATA CATGTGAAAT TCTTCACCAA GGACATATTA 10201TTGTGTCCAG AGGAAATCAG ACATGCTTGC AAGTTCGGGA TTGCTAAGGA TAATAATAAA 10261GACATGAGCT ATCCCCCTTG GGGAAGGGAA TCCAGAGGGA CAATTACAAC AATCCCTGTT 10321TATTATACGA CCACCCCTTA CCCAAAGATG CTAGAGATGC CTCCAAGAAT CCAAAATCCC 10381CTGCTGTCCG GAATCAGGTT GGGCCAATTA CCAACTGGCG CTCATTATAA AATTCGGAGT 10441ATATTACATG GAATGGGAAT CCATTACAGG GACTTCTTGA GTTGTGGAGA CGGCTCCGGA 10501GGGATGACTG CTGCATTACT ACGAGAAAAT GTGCATAGCA GAGGAATATT CAATAGTCTG 10561TTAGAATTAT CAGGGTCAGT CATGCGAGGC GCCTCTCCTG AGCCCCCCAG TGCCCTAGAA 10621ACTTTAGGAG GAGATAAATC GAGATGTGTA AATGGTGAAA CATGTTGGGA ATATCCATCT 10681GACTTATGTG ACCCAAGGAC TTGGGACTAT TTCCTCCGAC TCAAAGCAGG CTTGGGGCTT 10741CAAATTGATT TAATTGTAAT GGATATGGAA GTTCGGGATT CTTCTACTAG CCTGAAAATT 10801GAGACGAATG TTAGAAATTA TGTGCACCGG ATTTTGGATG AGCAAGGAGT TTTAATCTAC 10861AAGACTTATG GAACATATAT TTGTGAGAGC GAAAAGAATG CAGTAACAAT CCTTGGTCCC 10921ATGTTCAAGA CGGTCGACTT AGTTCAAACA GAATTTAGTA GTTCTCAAAC GTCTGAAGTA 10981TATATGGTAT GTAAAGGTTT GAAGAAATTA ATCGATGAAC CCAATCCCGA TTGGTCTTCC 11041ATCAATGAAT CCTGGAAAAA CCTGTACGCA TTCCAGTCAT CAGAACAGGA ATTTGCCAGA 11101GCAAAGAAGG TTAGTACATA CTTTACCTTG ACAGGTATTC CCTCCCAATT CATTCCTGAT 11161CCTTTTGTAA ACATTGAGAC TATGCTACAA ATATTCGGAG TACCCACGGG TGTGTCTCAT 11221GCGGCTGCCT TAAAATCATC TGATAGACCT GCAGATTTAT TGACCATTAG CCTTTTTTAT 11281ATGGCGATTA TATCGTATTA TAACATCAAT CATATCAGAG TAGGACCGAT ACCTCCGAAC 11341CCCCCATCAG ATGGAATTGC ACAAAATGTG GGGATCGCTA TAACTGGTAT AAGCTTTTGG 11401CTGAGTTTGA TGGAGAAAGA CATTCCACTA TATCAACAGT GTTTAGCAGT TATCCAGCAA 11461TCATTCCCGA TTAGGTGGGA GGCTGTTTCA GTAAAAGGAG GATACAAGCA GAAGTGGAGT 11521ACTAGAGGTG ATGGGCTCCC AAAAGATACC CGAATTTCAG ACTCCTTGGC CCCAATCGGG 11581AACTGGATCA GATCTCTGGA ATTGGTCCGA AACCAAGTTC GTCTAAATCC ATTCAATGAG 11641ATCTTGTTCA ATCAGCTATG TCGTACAGTG GATAATCATT TGAAATGGTC AAATTTGCGA 11701AAAAACACAG GAATGATTGA ATGGATCAAT AGACGAATTT CAAAAGAAGA CCGGTCTATA 11761CTGATGTTGA AGAGTGACCT ACACGAGGAA AACTCTTGGA GAGATTAAAA AATCATGAGG 11821AGACTCCAAA CTTTAAGTAT GAAAAAAACT TTGATCCTTA AGACCCTCTT GTGGTTTTTA 11881TTTTTTATCT GGTTTTGTGG TCTTCGTggc cggcatggtc ccagcctcct cgctggcgcc 11941ggctgggcaa cattccgagg ggaccgtccc ctcggtaatg gcgaatggga cctgctaaca 12001aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc 12061ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat 12121gcggccgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 12181gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 12241aaaggaggaa ctatatccgg gttaacctgc attaatgaat cggccaacgc gcggggagag 12301gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 12361ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 12421caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 12481aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 12541atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 12601cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 12661ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca 12721gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 12781accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 12841cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 12901cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 12961gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 13021aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 13081aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 13141actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 13201taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 13261gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 13321tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 13381ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 13441accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 13501agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 13561acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 13621tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 13681cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 13741tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 13801ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 13861gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 13921tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 13981ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 14041gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 14101cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 14161gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 14221ttccgcgcac atttccccga aaagtgccac ctgacgtc

An annotated sequence of SEQ ID NO 1 is provided below. The codedprotein is disclosed as SEQ ID NO: 2.

                                >T7-g10 Promoter                                |aa att aat acg act cac tat agg gag acc aca acg gtt tcc ctc tag cgt tgt ctt cgt c< 60             10           20            30           40           50                        >Hammerhead Ribozyme                        |tg atg agt ccg tga gga cga aac tat agg aaa gga att cct ata gtc ACG AAG ACA AAC A< 120             70           80            90           100          110                         >VSV Leader                         |AA CCA TTA TTA TCA TTA AAA GGC TCA GGA GAA ACT TTA ACA GTA ATC AAA ATG TCT GTT A< 180             130          140           150          160          170 CA GTC AAG AGA ATC ATT GAC AAC ACA GTC ATA GTT CCA AAA CTT CCT GCA AAT GAG GAT C< 240             190          200           210          220          230 CA GTG GAA TAC CCG GCA GAT TAC TTC AGA AAA TCA AAG GAG ATT CCT CTT TAC ATC AAT A< 300             250          260           270          280          290 CT ACA AAA AGT TTG TCA GAT CTA AGA GGA TAT GTC TAC CAA GGC CTC AAA TCC GGA AAT G< 360             810          320           330          340          350 TA TCA ATC ATA CAT GTC AAC AGC TAC TTG TAT GGA GCA TTG AAG GAC ATC CGG GGT AAG T< 420             370          380           390          400          410 TG GAT AAA GAT TGG TCA AGT TTC GGA ATA AAC ATC GGG AAG GCA GGG GAT ACA ATC GGA A< 480             430          440           450          460          470 TA TTT GAC CTT GTA TCC TTG AAA GCC CTG GAC GGT GTA CTT CCA GAT GGA GTA TCG GAT G< 540             490          500           510          520          530 CT TCC AGA ACC AGC GCA GAT GAC AAA TGG TTG CCT TTG TAT CTA CTT GGC TTA TAC AGA G< 600             550          560           570          580          590 TG GGC AGA ACA CAA ATG CCT GAA TAC AGA AAA AGG CTC ATG GAT GGG CTG ACA AAT CAA T< 660             610          620           630          640          650 GC AAA ATG ATC AAT GAA CAG TTT GAA CCT CTT GTG CCA GAA GGT CGT GAC ATT TTT GAT G< 720             670          680           690          700          710 TG TGG GGA AAT GAC AGT AAT TAC ACA AAA ATT GTC GCT GCA GTG GAC ATG TTC TTC CAC A< 780             730          740           750          760          770                                 >N                                 |TG TTC AAA AAA CAT GAA TGT GCC TCG TTC AGA TAC GGA ACT ATT GTT TCC AGA TTC AAA G< 840             790          800           810          820          830 AT TGT GCT GCA TTG GCA ACA TTT GGA CAC CTC TGC AAA ATA ACC GGA ATG TCT ACA GAA G< 900             850          860           870          880          890 AT GTG ACG ACC TGG AT TTG AAC CGA GAA GTT GCA GAT GAG AT GTC CAA ATG ATG CTT C< 960             910          920           930          940          950 CA GGC CAA GAA ATT GAC AAG GCT GAT TCA TAC ATG CCT TAT TTG ATC GAC TTT GGA TTG T< 1020             970          980           990          1000          1010 CT TCT AAG TCT CCA TAT TCT TCC GTC AAA AAC CCT GCC TTC CAC TTC TGG GGG CAA TTG A< 1080             1030         1040          1050         1060         1070 CA GCT CTT CTG CTC AGA TCC ACC AGA GCA AGG AAT GCC CGA CAG CCT GAT GAC ATT GAG T< 1140             1090         1100          1110         1120         1130 AT ACA TCT CTT ACT ACA GCA GGT TTG TTG TAC GCT TAT GCA GTA GGA TCC TCT GCT GAC T< 1200             1150         1160          1170         1180         1190 TG GCA CAA CAG TTT TGT GTT GGA GAT AGC AAA TAC ACT CCA GAT GAT AGT ACC GGA GGA T< 1260             1210         1220          1230         1240         1250 TG ACG ACT AAT GCA CCG CCA CAA GGC AGA GAT GTG GTC GAA TGG CTC GGA TGG TTT GAA G< 1320             1270         1280          1290         1300         1310 AT CAA AAC AGA AAA CCG ACT CCT GAT ATG ATG CAG TAT GCG AAA CGA GCA GTC ATG TCA C< 1380             1330         1340          1350         1360         1370 TG CAA GGC CTA AGA GAG AAG ACA ATT GGC AAG TAT GCT AAG TCA GAG TTT GAC AAA TGA C< 1440             1390         1400          1410         1420         1430 CC TAT AAT TCT CAG ATC ACC TAT TAT ATA TTA TGC TAG CTA TGA AAA AAA CTA ACA GAT A< 1500             1450         1460          1470         1480         1490 TC ATG GAT AAT GTG AGA AAA GTT GCT GAG TAT GTG AAG TCG TAT TCT GCT GTA GAT GAG G< 1560             1510         1520          1530         1540         1550 CG GTA GGA GAG ATA GAT GAG ATC GAA GCA CAA CGA GCT GAA AAG TCC AAT TAT GAG TTG T< 1620             1570         1580          1590         1600         1610 TC CAA GAG GAC GGA GTG GAA GAG CAT ACT AGG CCC TCT TAT TTT CAG GCA GCA GAT GAT T< 1680             1630         1640          1650         1660         1670 CT GAC AGA GAA TCT GAA CCA GAA ATT GAA GAC AAT CAA GGC TTG TAT GTA CCA GAT CCG G< 1740             1690         1700          1710         1720         1730 AA GCT GAG CAA GTT GAA GGC TTT ATA CAG GGG CCT TTA GAT GAC TAT GCA GAT GAG GAC G< 1800             1750         1760          1770         1780         1790 TG GAT GTT GTA TTG ACT TCG GAC TGG AAA CAG CCT GAG CTT GAA TCC GAG GAG GAT GGA A< 1860             1810         1820          1830         1840         1850                                                      >P                                                     |AG ACC TTA CGG TTG ACA TTG CCA GAG GGT TTA AGT GGA GAG CAG AAA TCC CAG TGG CTT T< 1920             1870         1880          1890         1900         1910 TG ACG ATT AAA GCA GTC GTT CAA AGT GCC AAA CAC TGG AAT CTG GCA GAG TGC ACA TTT G< 1980             1930         1940          1950         1960         1970 AA GCA TCG GGA GAA GGG GTC ATC ATA AAA AAG CGC CAG ATA ACT CCG GAT GTA TAT AAG G< 2040             1990         2000          2010         2020         2030 TC ACT CCA GTG ATG AAC ACA CAT CCG TCC CAA TCA GAA GCC GTA TCA GAT GTT TGG TCT C< 2100             2050         2060          2070         2080         2090 TC TCA AAG ACA TCC ATG ACT TTC CAA CCC AAG AAA GCA AGT CTT CAG CCT CTC ACC ATA T< 2160             2110         2120          2130         2140         2150 CC TTG GAT GAA TTG TTC TCA TCT AGA GGA GAA TTC ATC TCT GTC GGA GGT AAC GGA CGA A< 2220             2170         2180          2190         2200         2210 TG TCT CAT AAA GAG GCC ATC CTG CTC GGT CTG AGG TAC AAA AAG TTG TAC AAT CAG GCG A< 2280             2230         2240          2250         2260         2270 GA GTC AAA TAT TCT CTG TAG ACT AGT ATG AAA AAA AGT AAC AGA TAT CAC AAT CTA AGT G< 2340             2290         2300          2310         2320         2330 TT ATC CCA ATC CAT TCA TCA TGA GTT CCT TAA AGA AGA TTC TCG GTC TGA AGG GGA AAG G< 2400             2350         2360          2370         2380         2390 TA AGA AAT CTA AGA AAT TAG GGA TCG CAC CAC CCC CTT ATG AAG AGG ACA CTA ACA TGG A< 2460             2410         2420          2430         2440         2450 GT ATG CTC CGA GCG CTC CAA TTG ACA AAT CCT ATT TTG GAG TTG ACG AGA TGG ACA CTC A< 2520             2470         2480          2490         2500         2510 TG ATC CGA ATC AAT TAA GAT ATG AGA AAT TCT TCT TTA CAG TGA AAA TGA CGG TTA GAT C< 2580             2530         2540          2550         2560         2570 TA ATC GTC CGT TCA GAA CAT ACT CAG ATG TGG CAG CCG CTG TAT CCC ATT GGG ATC ACA T< 2640             2590         2600          2610         2620         2630 GT ACA TCG GAA TGG CAG GGA AAC GTC CCT TCT ACA AGA TCT TGG CTT TTT TGG GTT CTT C< 2700             2650         2660          2670         2680         2690     >M     |TA ATC TAA AGG CCA CTC CAG CGG TAT TGG CAG ATC AAG GTC AAC CAG AGT ATC ATG CTC A< 2760             2710         2720          2730         2740         2750 CT GTG AAG GCA GGG CTT ATT TGC CAC ACA GAA TGG GGA AGA CCC CTC CCA TGC TCA ATG T< 2820             2770         2780          2790         2800         2810 AC CAG AGC ACT TCA GAA GAC CAT TCA ATA TAG GTC TTT ACA AGG GAA CGA TTG AGC TCA C< 2880             2830         2840          2850         2860         2870 AA TGA CCA TCT ACG ATG ATG AGT CAC TGG AAG CAG CTC CTA TGA TCT GGG ATC ATT TCA A< 2940             2890         2900          2910         2920         2930 TT CTT CCA AAT TTT CTG ATT TCA GAG AGA AGG CCT TAA TGT TTG GCC TGA TTG TCG AGA A< 3000             2950         2960          2970         2980         2990 AA AGG CAT CTG GAG CTT GGG TCC TGG ATT CTG TCA GCC ACT TCA AAT GAG CTA GTC TAG C< 3060             3010         3020          3030         3040         3050 TT CCA GCT TCT GAA CAA TCC CCG GTT TAC TCA GTC TCT CCT AAT TCC AGC CTT TCG AAC A< 3120             3070         3080          3090         3100         3110 AC TAA TAT CCT GTC TTC TCT ATC CCT ATG AAA AAA ACT AAC AGA GAT CGA TCT GTT TCC T< 3180             3130         3140          3150         3160         3170 TG ACA CCA GGA GCC ACC ATG AAG TGC CTT TTG TAC TTA GCT TTT TTA TTC ATC GGG GTG A < 3240                        M   K   C   L   L   Y   L   A   F   L   F   I   G   V   N            3190         3200          3210         3220         3230 AT TGC AAG GCT AGC GCA GAG AAT TTG TGG GTA ACA GTC TAC TAT GGA GTC CCT GTA TGG A< 3300    C   K   A   S   A   E   N   L   W   V   T   V   Y   Y   G   V   P   V   W   K            3250         3260          3270         3280         3290 AG GAT GCA GAG ACA ACA TTG TTC TGT GCT AGT GAC GCA AAG GCT TAC GAG ACG GAG AAG C< 3360    D   A   E   T   T   L   F   C   A   S   D   A   K   A   Y   E   T   E   K   H            3310         3320          3330         3340         3350 AC AAT GTG TGG GCA ACT CAC GCA TGT GTC CCA ACC GAT CCA AAT CCT CAA GAG ATT CAT C< 3420    N   V   W   A   T   H   A   C   V   P   T   D   P   N   P   Q   E   I   H   L            3370         3380          3390         3400         3410 TA GAG AAT GTG ACT GAA GAA TTC AAT ATG TGG AAG AAT AAT ATG GTA GAG CAA ATG CAT A< 3480    E   N   V   T   E   E   F   N   M   W   K   N   N   M   V   E   Q   M   H   T            3430         3440          3450         3460         3470 CA GAT ATC ATT AGT TTA TGG GAC CAG TCA CTT AAA CCC TGC GTT AAA TTG ACG CCT CTA T< 3540    D   I   I   S   L   W   D   Q   S   L   K   P   C   V   K   L   T   P   L   C            3490         3500          3510         3520         3530 GT GTG ACA CTT CAA TGT ACT AAT GTT ACA AAC AAC ATA ACA GAT GAT ATG AGA GGA GAA C< 3600    V   T   L   Q   C   T   N   V   T   N   N   I   T   D   D   M   R   G   E   L            3550         3560          3570         3580         3590 TG AAG AAC TGT AGT TTC AAC ATG ACG ACA GAG TTG CGT GAC AAG AAA CAG AAA GTG TAT T< 3660    K   N   C   S   F   N   M   T   T   E   L   R   D   K   K   Q   K   V   Y   S            3610         3620          3630         3640         3650 CA CTA TTC TAT CGG TTG GAT GTA GTA CAG ATA AAT GAG AAT CAA GGA AAC AGG TCC AAC A< 3720    L   F   Y   R   L   D   V   V   Q   I   N   E   N   Q   G   N   R   S   N   N            3670         3680          3690         3700         3710 AC TCT AAC AAA GAG TAC AGA CTT AGG AAT TGC AAT ACC AGT GCT ATC ACG CAA GCC TGC C< 3780    S   N   K   E   Y   R   L   I   N   C   N   T   S   A   I   T   Q   A   C   P            3730         3740          3750         3760         3770 CA AAG GTT TCA TTT GAA CCA ATA CCT ATT CAT TAT TGT GCA CCT GCT GGA TTC GCC ATC C< 3840    K   V   S   F   E   P   I   P   I   H   Y   C   A   P   A   G   F   A   I   L            3790         3800          3810         3820         3830 TC AAA TGT AAA GAC AAG AAG TTC AAT GGA ACA GGA CCC TGC CCA TCA GTT TCA ACC GTT C< 3900    K   C   K   D   K   K   F   N   G   T   G   P   C   P   S   V   S   T   V   Q            3850         3860          3870         3880         3890 AG TGC ACC CAC GGA ATC AAG CCT GTA GTT AGT ACT CAA TTA TTG TTA AAT GGG AGC TTA G< 3960    C   T   H   G   I   K   P   V   V   S   T   Q   L   L   L   N   G   S   L   A            3910         3920          3930         3940         3950 CT GAA GAA GAA GTT ATG ATT AGA TCA GAG AAT ATT ACC AAT AAT GCG AAG AAC ATC TTG G< 4020    E   E   E   V   M   I   R   S   E   N   I   T   N   N   A   K   N   I   L   V            3970         3980          3990         4000         4010 TT CAA TTC AAT ACT CCA GTC CAG ATC AAT TGC ACA AGG CCT AAT AAT AAT ACC AGA AAG A< 4080    Q   F   N   T   P   V   Q   I   N   C   T   R   P   N   N   N   T   R   K   S            4030         4040          4050         4060         4070 GT ATA AGA ATT GGG CCA GGA CAG GCA TTC TAT GCA ACA GGA GAT ATA ATC GGA GAC ATT C< 4140    I   R   I   G   P   G   Q   A   F   Y   A   T   G   D   I   I   G   D   I   R            4090         4100          4110         4120         4130 GA CAA GCG CAC TGC ACT GTT TCT AAG GCC ACT TGG AAT GAA ACA TTG GGT AAA GTT GTA A< 4200    Q   A   H   C   T   V   S   K   A   T   W   N   E   T   L   G   K   V   V   K            4150         4160          4170         4180         4190 AG CAA CTT CGG AAG CAT TTC GGA AAT AAC ACA ATT ATT AGA TTT GCG AAC TCA TCT GGA G< 4260    Q   L   R   K   H   F   G   N   N   T   I   I   R   F   A   N   S   S   G   G            4210         4220          4230         4240         4250                      >Env.BG505 immunogen                      |GG GAT CTG GAA GTG ACA ACA CAC TCT TTC AAT TGC GGT GGC GAG TTC TTC TAT TGT AAT A< 4320    D   L   E   V   T   T   H   S   F   N   C   G   G   E   F   F   Y   C   N   T            4270         4280          4290         4300         4310 CA AGT GGA TTA TTT AAC TCT ACT TGG ATT TCA AAT ACC TCA GTC CAA GGA TCT AAT TCA A< 4380    S   G   L   F   N   S   T   W   I   S   N   T   S   V   Q   G   S   N   S   T            4330         4340          4350         4360         4370 CA GGG TCT AAC GAT TCT ATA ACA TTA CCT TGC CGT ATA AAG CAA ATT ATT AAT ATG TGG C< 4440    G   S   N   D   S   I   T   L   P   C   R   I   K   Q   I   I   N   M   W   Q            4390         4400          4410         4420         4430 AA AGA ATC GGG CAA GCG ATG TAT GCT CCA CCT ATT CAA GGC GTG ATT CGT TGC GTT TCA A< 4500    R   I   G   Q   A   M   Y   A   P   P   I   Q   G   V   I   R   C   V   S   N            4450         4460          4470         4480         4490 AC ATA ACA GGG TTG ATC CTG ACC AGG GAT GGA GGC TCT ACC AAT TCC ACC ACC GAG ACC T< 4560    I   T   G   L   I   L   T   R   D   G   G   S   T   N   S   T   T   E   T   F            4510         4520          4530         4540         4550 TC CGT CCC GGT GGC GGA GAT ATG CGG GAT AAC TGG AGA TCA GAG CTC TAT AAG TAT AAG G< 4620    R   P   G   G   G   D   M   R   D   N   W   R   S   E   L   Y   K   Y   K   V            4570         4580          4590         4600         4610 TT GTG AAG ATT GAA CCT CTT GGA GTT GCC CCT ACA AGA GCA AAG AGA AGG GTG GTT GGC C< 4680    V   K   I   E   P   L   G   V   A   P   T   R   A   K   R   R   V   V   G   R            4630         4640          4650         4660         4670 GA GAG AAG AGA GCA GTT GGC ATC GGT GCT GTC TTT CTC GGA TTT CTT GGA GCA GCT GGA T< 4740    E   K   R   A   V   G   I   G   A   V   F   L   G   F   L   G   A   A   G   S            4690         4700          4710         4720         4730 CC ACT ATG GGA GCA GCA TCA ATG ACA CTA ACA GTG CAG GCT AGA AAT TTG CTT AGC GGA A< 4800    T   M   G   A   A   S   M   T   L   T   V   Q   A   R   N   L   L   S   G   I            4750         4760          4770         4780         4790 TC GTT CAG CAG CAG AGC AAT TTA CTA AGA GCA ATT GAA GCA CAG CAA CAT CTC TTA AAG T< 4860    V   Q   Q   Q   S   N   L   L   R   A   I   E   A   Q   Q   H   L   L   K   L            4810         4820          4830         4840         4850 TG ACG GTG TGG GGC ATT AAA CAA CTA CAA GCT AGA GTG CTT GCC GTC GAA AGA TAT TTG C< 4920    T   V   W   G   I   K   Q   L   Q   A   R   V   L   A   V   E   R   Y   L   R            4870         4880          4890         4900         4910 GA GAC CAA CAG CTA TTG GGT ATT TGG GGT TGT TCT GGG AAA TTA ATT TGC ACA ACA AAT G< 4980    D   Q   Q   L   L   G   I   W   G   C   S   G   K   L   I   C   T   T   N   V            4930         4940          4950         4960         4970 TT CCA TGG AAC TCC TCC TGG AGT AAT AGG AAT TTA AGT GAG ATA TGG GAC AAC ATG ACA T< 5040    P   W   N   S   S   W   S   N   R   N   L   S   E   I   W   D   N   M   T   W            4990         5000          5010         5020         5030 GG TTG CAG TGG GAC AAG GAA ATC TCA AAT TAT ACA CAG ATA ATC TAT GGA TTA TTA GAA G< 5100    L   Q   W   D   K   E   I   S   N   Y   T   Q   I   I   Y   G   L   L   E   E            5050         5060          5070         5080         5090 AG TCT CAG AAT CAG CAA GAG AAG AAT GAA CAG GAT TTG CTT GCA TTG GAT AAG TGG GCT T< 5160    S   Q   N   Q   Q   E   K   N   E   Q   D   L   L   A   L   D   K   W   A   S            5110         5120          5130         5140         5150 CT CTA TGG AAC TGG TTC GAT ATT AGT AAT TGG CTC TGG TAT ATT AAG AGC TCT ATT GCC T< 5220    L   W   N   W   F   D   I   S   N   W   L   W   Y   I   K   S   S   I   A   S            5170         5180          5190         5200         5210 CT TTT TTC TTT ATC ATA GGG TTA ATC ATT GGA CTA TTC TTG GTT CTC CGA GTT GGT ATT T< 5280    F   F   F   I   I   G   L   I   I   G   L   F   L   V   L   R   V   G   I   Y            5230         5240          5250         5260         5270 AT CTT TGC ATT AAA TTA AAG CAC ACC AAG AAA AGA CAG ATT TAT ACA GAC ATA GAG ATG A< 5340    L   C   I   K   L   K   H   T   K   K   R   Q   I   Y   T   D   I   E   M   N            5290         5300          5310         5320         5330 AC CGA CTT GGA AAG TAA AGC TCA AAT CCT GCA CAA CAG ATT CTT CAT GTT TGA ACC AAA T< 5400     R   L   G   K   *            5350         5360          5370         5380         5390 CA ACT TGT GAT ATC ATG CTC AAA GAG GCC TTA ATT AAA TTT TAA TTT TTA ATT TTT ATG A< 5460             5410         5420          5430         5440         5450 AA AAA ACT AAC AGC AAT CAT GGA AGT CCA CGA TTT TGA GAC CGA CGA GTT CAA TGA TTT C< 5520             5470         5480          5490         5500         5510 AA TGA AGA TGA CTA TGC CAC AAG AGA ATT CCT GAA TCC CGA TGA GCG CAT GAC GTA CTT G< 5580             5530         5540          5550         5560         5570 AA TCA TGC TGA TTA CAA TTT GAA TTC TCC TCT AAT TAG TGA TGA TAT TGA CAA TTT GAT C< 5640             5590         5600          5610         5620         5630 AG GAA ATT CAA TTC TCT TCC GAT TCC CTC GAT GTG GGA TAG TAA GAA CTG GGA TGG AGT T< 5700             5650         5660          5670         5680         5690 CT TGA GAT GTT AAC ATC ATG TCA AGC CAA TCC CAT CTC AAC ATC TCA GAT GCA TAA ATG G< 5760             5710         5720          5730         5740         5750 AT GGG AAG TTG GTT AAT GTC TGA TAA TCA TGA TGC CAG TCA AGG GTA TAG TTT TTT ACA T< 5820             5770         5780          5790         5800         5810 GA AGT GGA CAA AGA GGC AGA AAT AAC ATT TGA CGT GGT GGA GAC CTT CAT CCG CGG CTG G< 5880             5830         5840          5850         5860         5870 GG CAA CAA ACC AAT TGA ATA CAT CAA AAA GGA AAG ATG GAC TGA CTC ATT CAA AAT TCT C< 5940             5890         5900          5910         5920         5930 GC TTA TTT GTG TCA AAA GTT TTT GGA CTT ACA CAA GTT GAC ATT AAT CTT AAA TGC TGT C< 6000             5950         5960          5970         5990         5990 TC TGA GGT GGA ATT GCT CAA CTT GGC GAG GAC TTT CAA AGG CAA AGT CAG AAG AAG TTC T< 6060             6010         6020          6030         6040         6050 CA TGG AAC GAA CAT ATG CAG GCT TAG GGT TCC CAG CTT GGG TCC TAC TTT TAT TTC AGA A< 6120             6070         6080          6090         6100         6110 GG ATG GGC TTA CTT CAA GAA ACT TGA TAT TCT AAT GGA CCG AAA CTT TCT GTT AAT GGT C< 6180             6130         6140          6150         6160         6170 AA AGA TGT GAT TAT AGG GAG GAT GCA AAC GGT GCT ATC CAT GGT ATG TAG AAT AGA CAA C< 6240             6190         6200          6210         6220         6230 CT GTT CTC AGA GCA AGA CAT CTT CTC CCT TCT AAA TAT CTA CAG AAT TGG AGA TAA AAT T< 6300             6250         6260          6270         6290         6290 GT GGA GAG GCA GGG AAA TTT TTC TTA TGA CTT GAT TAA AAT GGT GGA ACC GAT ATG CAA C< 6360             6310         6320          6330         6340         6350 TT GAA GCT GAT GAA ATT AGC AAG AGA ATC AAG GCC TTT AGT CCC ACA ATT CCC TCA TTT T< 6420             6370         6380          6390         6400         6410 GA AAA TCA TAT CAA GAC TTC TGT TGA TGA AGG GGC AAA AAT TGA CCG AGG TAT AAG ATT C< 6480             6430         6440          6450         6460         6470 CT CCA TGA TCA GAT AAT GAG TGT GAA AAC AGT GGA TCT CAC ACT GGT GAT TTA TGG ATC G< 6540             6490         6500          6510         6520         6530 TT CAG ACA TTG GGG TCA TCC TTT TAT AGA TTA TTA CGC TGG ACT AGA AAA ATT ACA TTC C< 6600             6550         6560          6570         6580         6590 CA AGT AAC CAT GAA GAA AGA TAT TGA TGT GTC ATA TGC AAA AGC ACT TGC AAG TGA TTT A< 6660             6610         6620          6630         6640         6650 GC TCG GAT TGT TCT ATT TCA ACA GTT CAA TGA TCA TAA AAA GTG GTT CGT GAA TGG AGA C< 6720             6670         6680          6690         6700         6710 TT GCT CCC TCA TGA TCA TCC CTT TAA AAG TCA TGT TAA AGA AAA TAC ATG GCC TAC AGC T< 6780             6730         6740          6750         6760         6770 GC TCA AGT TCA AGA TTT TGG AGA TAA ATG GCA TGA ACT TCC GCT GAT TAA ATG TTT TGA A< 6840             6790         6900          6910         6820         6930 AT ACC CGA CTT ACT AGA CCC ATC GAT AAT ATA CTC TGA CAA AAG TCA TTC AAT GAA TAG G< 6900             6850         6860          6870         6880         6890 TC AGA GGT GTT GAA ACA TGT CCG AAT GAA TCC GAA CAC TCC TAT CCC TAG TAA AAA GGT G< 6960             6910         6920          6930         6940         6950 TT GCA GAC TAT GTT GGA CAC AAA GGC TAC CAA TTG GAA AGA ATT TCT TAA AGA GAT TGA T< 7020             6970         6980          6990         7000         7010 GA GAA GGG CTT AGA TGA TGA TGA TCT AAT TAT TGG TCT TAA AGG AAA GGA GAG GGA ACT G< 7080             7030         7040          7050         7060         7070 AA GTT GGC AGG TAG ATT TTT CTC CCT AAT GTC TTG GAA ATT GCG AGA ATA CTT TGT AAT T< 7140             7090         7100          7110         7120         7130 AC CGA ATA TTT GAT AAA GAC TCA TTT CGT CCC TAT GTT TAA AGG CCT GAC AAT GGC GGA C< 7200             7150         7160          7170         7180         7190 GA TCT AAC TGC AGT CAT TAA AAA GAT GTT AGA TTC CTC ATC CGG CCA AGG ATT GAA GTC A< 7260             7210         7220          7230         7240         7250 TA TGA GGC AAT TTG CAT AGC CAA TCA CAT TGA TTA CGA AAA ATG GAA TAA CCA CCA AAG G< 7320             7270         7280          7290         7300         7310 AA GTT ATC AAA CGG CCC AGT GTT CCG AGT TAT GGG CCA GTT CTT AGG TTA TCC ATC CTT A< 7380             7330         7340          7350         7360         7370 AT CGA GAG AAC TCA TGA ATT TTT TGA GAA AAG TCT TAT ATA CTA CAA TGG AAG ACC AGA C< 7440             7390         7400          7410         7420         7430 TT GAT GCG TGT TCA CAA CAA CAC ACT GAT CAA TTC AAC CTC CCA ACG AGT TTG TTG GCA A< 7500             7450         7460          7470         7480         7490 GG ACA AGA GGG TGG ACT GGA AGG TCT ACG GCA AAA AGG ATG GAG TAT CCT CAA TCT ACT G< 7560             7510         7520          7530         7540         7550 GT TAT TCA AAG AGA GGC TAA AAT CAG AAA CAC TGC TGT CAA AGT CTT GGC ACA AGG TGA T< 7620             7570         7580          7590         7600         7610 AA TCA AGT TAT TTG CAC ACA GTA TAA AAC GAA GAA ATC GAG AAA CGT TGT AGA ATT ACA G< 7680             7630         7640          7650         7660         7670 GG TGC TCT CAA TCA AAT GGT TTC TAA TAA TGA GAA AAT TAT GAC TGC AAT CAA AAT AGG G< 7740             7690         7700          7710         7720         7730 AC AGG GAA GTT AGG ACT TTT GAT AAA TGA CGA TGA GAC TAT GCA ATC TGC AGA TTA CTT G< 7800             7750         7760          7770         7780         7790 AA TTA TGG AAA AAT ACC GAT TTT CCG TGG AGT GAT TAG AGG GTT AGA GAC CAA GAG ATG G< 7860             7810         7820          7830         7840         7850 TC ACG AGT GAC TTG TGT CAC CAA TGA CCA AAT ACC CAC TTG TGC TAA TAT AAT GAG CTC A< 7920             7870         7880          7890         7900         7910 GT TTC CAC AAA TGC TCT CAC CGT AGC TCA TTT TGC TGA GAA CCC AAT CAA TGC CAT GAT A< 7980             7930         7940          7950         7960         7970 CA GTA CAA TTA TTT TGG GAC ATT TGC TAG ACT CTT GTT GAT GAT GCA TGA TCC TGC TCT T< 8040             7990         8000          8010         8020         8030 CG TCA ATC ATT GTA TGA AGT TCA AGA TAA GAT ACC GGG CTT GCA CAG TTC TAC TTT CAA A< 8100             8050         8060          8070         8080         8090 TA CGC CAT GTT GTA TTT GGA CCC TTC CAT TGG AGG AGT GTC GGG CAT GTC TTT GTC CAG G< 8160             8110         8120          8130         8140         8150 TT TTT GAT TAG AGC CTT CCC AGA TCC CGT AAC AGA AAG TCT CTC ATT CTG GAG ATT CAT C< 8220             8170         8180          8190         8200         8210 CA TGT ACA TGC TCG AAG TGA GCA TCT GAA GGA GAT GAG TGC AGT ATT TGG AAA CCC CGA G< 8280             8230         8240          8250         8260         8270 AT AGC CAA GTT CCG AAT AAC TCA CAT AGA CAA GCT AGT AGA AGA TCC AAC CTC TCT GAA C< 8340             8290         8300          8310         8320         8330 AT CGC TAT GGG AAT GAG TCC AGC GAA CTT GTT AAA GAC TGA GGT TAA AAA ATG CTT AAT C< 8400             8350         8360          8370         8380         8390 GA ATC AAG ACA AAC CAT CAG GAA CCA GGT GAT TAA GGA TGC AAC CAT ATA TTT GTA TCA T< 8460             8410         8420          8430         8440         8450 GA AGA GGA TCG GCT CAG AAG TTT CTT ATG GTC AAT AAA TCC TCT GTT CCC TAG ATT TTT A< 8520             8470         8480          8490         8500         8510 AG TGA ATT CAA ATC AGG CAC TTT TTT GGG AGT CGC AGA CGG GCT CAT CAG TCT ATT TCA A< 8580             8530         8540          8550         8560         8570 AA TTC TCG TAC TAT TCG GAA CTC CTT TAA GAA AAA GTA TCA TAG GGA ATT GGA TGA TTT G< 8640             8590         8600          8610         8620         8630    >L    |AT TGT GAG GAG TGA GGT ATC CTC TTT GAC ACA TTT AGG GAA ACT TCA TTT GAG AAG GGG A< 8700             8650         8660          8670         8680         8690 TC ATG TAA AAT GTG GAC ATG TTC AGC TAC TCA TGC TGA CAC ATT AAG ATA CAA ATC CTG G< 8760             8710         8720          8730         8740         8750 GG CCG TAC AGT TAT TGG GAC AAG TGT ACC CCA TCC ATT AGA AAT GTT GGG TCC ACA ACA T< 8820             8770         8780          8790         8800         8810 CG AAA AGA GAC TCC TTG TGC ACC ATG TAA CAC ATC AGG GTT CAA TTA TGT TTC TGT GCA T< 8880             8830         8840          8850         8860         8870 TG TCC AGA CGG GAT CCA TGA CGT CTT TAG TTC ACG GGG ACC ATT GCC TGC TTA TCT AGG G< 8940             8890         8900          8910         8920         8930 TC TAA AAG ATC TGA ATC TAC ATC TAT TTT GCA GCC TTG GGA AAG GGA AAG CAA AGT CCC A< 9000             8950         8960          8970         8980         8990 CT GAT TAA AAG AGC TAC ACG TCT TAG AGA TGC TAT CTC TTG GTT TGT TGA ACC CGA CTC T< 9060             9010         9020          9030         9040         9050 AA ACT AGC AAT GAC TAT ACT TTC TAA CAT CCA CTC TTT AAG AGG CGA AGA ATG GAC CAA A< 9120             9070         9080          9090         9100         9110 AG GCA GCA TGG GTT CAA AAG AAC AGG GTC TGC CCT TCA TAG GTT TTC GAC ATC TCG GAT G< 9180             9130         9140          9150         9160         9170 AG CCA TGG TGG GTT GGG ATC TCA GAG CAC TGC AGC ATT GAC CAG GTT GAT GGC AAG TAC A< 9240             9190         9200          9210         9220         9230 GA CAC CAT GAG GGA TCT GGG AGA TCA GAA TTT CGA CTT TTT ATT CCA AGC AAC GTT GCT C< 9300             9250         9260          9270         9280         9290 TA TGC TCA AAT TAC CAC CAC TGT TGC AAG AGA CGG ATG GAT CAC CAG TTG TAC AGA TCA T< 9360             9310         9320          9330         9340         9350 TA TCA TAT TGC CTG TAA GTC CTG TTT GAG ACC CAT AGA AGA GAT CAC CCT GGA CTC AAG T< 9420             9370         9380          9390         9400         9410 AT GGA CTA CAC GCC CCC AGA TGT ATC CCA TGT GCT GAA GAC ATG GAG GAA TGG GGA AGG T< 9480             9430         9440          9450         9460         9470 TC GTG GGG ACA AGA GAT AAA ACA GAT CTA TCC TTT AGA AGG GAA TTG GAA GAA TTT AGC A< 9540             9490         9500          9510         9520         9530 CC TGC TGA GCA ATC CTA TCA AGT CGG CAG ATG TAT AGG TTT TCT ATA TGG AGA CTT GGC G< 9600             9550         9560          9570         9580         9590 TA TAG AAA ATC TAC TCA TGC CGA GGA CAG TTC TCT ATT TCC TCT ATC TAT ACA AGG TCG T< 9660             9610         9620          9630         9640         9650 AT TAG AGG TCG AGG TTT CTT AAA AGG GTT GCT AGA CGG ATT AAT GAG AGC AAG TTG CTG C< 9720             9670         9680          9690         9700         9710 CA AGT AAT ACA CCG GAG AAG TCT GGC TCA TTT GAA GAG GCC GGC CAA CGC AGT GTA CGG A< 9780             9730         9740          9750         9760         9770 GG TTT GAT TTA CTT GAT TGA TAA ATT GAG TGT ATC ACC TCC ATT CCT TTC TCT TAC TAG A< 9840             9790         9800          9810         9820         9830 TC AGG ACC TAT TAG AGA CGA ATT AGA AAC GAT TCC CCA CAA GAT CCC AAC CTC CTA TCC G< 9900             9850         9860          9870         9880         9890 AC AAG CAA CCG TGA TAT GGG GGT GAT TGT CAG AAA TTA CTT CAA ATA CCA ATG CCG TCT A< 9960             9910         9920          9930         9940         9950 AT TGA AAA GGG AAA ATA CAG ATC ACA TTA TTC ACA ATT ATG GTT ATT CTC AGA TGT CTT A< 10020             9970         9980          9990         10000        10010 TC CAT AGA CTT CAT TGG ACC ATT CTC TAT TTC CAC CAC CCT CTT GCA AAT CCT ATA CAA G< 10080             10030        10040         10050        10060        10070 CC ATT TTT ATC TGG GAA AGA TAA GAA TGA GTT GAG AGA GCT GGC AAA TCT TTC TTC ATT G< 10140             10090        10100         10110        10120        10130 CT AAG ATC AGG AGA GGG GTG GGA AGA CAT ACA TGT GAA ATT CTT CAC CAA GGA CAT ATT A< 10200             10150        10160         10170        10180        10190 TT GTG TCC AGA GGA AAT CAG ACA TGC TTG CAA GTT CGG GAT TGC TAA GGA TAA TAA TAA A< 10260             10210        10220         10230        10240        10250 GA CAT GAG CTA TCC CCC TTG GGG AAG GGA ATC CAG AGG GAC AAT TAC AAC AAT CCC TGT T< 10320             10270        10280         10290        10300        10310 TA TTA TAC GAC CAC CCC TTA CCC AAA GAT GCT AGA GAT GCC TCC AAG AAT CCA AAA TCC C< 10380             10330        10340         10350        10360        10370 CT GCT GTC CGG AAT CAG GTT GGG CCA ATT ACC AAC TGG CGC TCA TTA TAA AAT TCG GAG T< 10440             10390        10400         10410        10420        10430 AT ATT ACA TGG AAT GGG AAT CCA TTA CAG GGA CTT CTT GAG TTG TGG AGA CGG CTC CGG A< 10500             10450        10460         10470        10480        10490 GG GAT GAC TGC TGC ATT ACT ACG AGA AAA TGT GCA TAG CAG AGG AAT ATT CAA TAG TCT G< 10560             10510        10520         10530        10540        10550 TT AGA ATT ATC AGG GTC AGT CAT GCG AGG CGC CTC TCC TGA GCC CCC CAG TGC CCT AGA A< 10620             10570        10580         10590        10600        10610 AC TTT AGG AGG AGA TAA ATC GAG ATG TGT AAA TGG TGA AAC ATG TTG GGA ATA TCC ATC T< 10680             10630        10640         10650        10660        10670 GA CTT ATG TGA CCC AAG GAC TTG GGA CTA TTT CCT CCG ACT CAA AGC AGG CTT GGG GCT T< 10740             10690        10700         10710        10720        10730 CA AAT TGA TTT AAT TGT AAT GGA TAT GGA AGT TCG GGA TTC TTC TAC TAG CCT GAA AAT T< 10800             10750        10760         10770        10780        10790 GA GAC GAA TGT TAG AAA TTA TGT GCA CCG GAT TTT GGA TGA GCA AGG AGT TTT AAT CTA C< 10860             10810        10820         10830        10840        10850 AA GAC TTA TGG AAC ATA TAT TTG TGA GAG CGA AAA GAA TGC AGT AAC AAT CCT TGG TCC C< 10920             10870        10880         10890        10900        10910 AT GTT CAA GAC GGT CGA CTT AGT TCA AAC AGA ATT TAG TAG TTC TCA AAC GTC TGA AGT A< 10980             10930        10940         10950        10960        10970 TA TAT GGT ATG TAA AGG TTT GAA GAA ATT AAT CGA TGA ACC CAA TCC CGA TTG GTC TTC C< 11040             10990        11000         11010        11020        11030 AT CAA TGA ATC CTG GAA AAA CCT GTA CGC ATT CCA GTC ATC AGA ACA GGA ATT TGC CAG A< 11100             11050        11060         11070        11080        11090 GC AAA GAA GGT TAG TAC ATA CTT TAC CTT GAC AGG TAT TCC CTC CCA ATT CAT TCC TGA T< 11160             11110        11120         11130        11140        11150 CC TTT TGT AAA CAT TGA GAC TAT GCT ACA AAT ATT CGG AGT ACC CAC GGG TGT GTC TCA T< 11220             11170        11180         11190        11200        11210 GC GGC TGC CTT AAA ATC ATC TGA TAG ACC TGC AGA TTT ATT GAC CAT TAG CCT TTT TTA T< 11280             11230        11240         11250        11260        11270 AT GGC GAT TAT ATC GTA TTA TAA CAT CAA TCA TAT CAG AGT AGG ACC GAT ACC TCC GAA C< 11340             11290        11300         11310        11320        11330 CC CCC ATC AGA TGG AAT TGC ACA AAA TGT GGG GAT CGC TAT AAC TGG TAT AAG CTT TTG G< 11400             11350        11360         11370        11380        11390 CT GAG TTT GAT GGA GAA AGA CAT TCC ACT ATA TCA ACA GTG TTT AGC AGT TAT CCA GCA A< 11460             11410        11420         11430        11440        11450 TC ATT CCC GAT TAG GTG GGA GGC TGT TTC AGT AAA AGG AGG ATA CAA GCA GAA GTG GAG T< 11520             11470        11480         11490        11500        11510 AC TAG AGG TGA TGG GCT CCC AAA AGA TAC CCG AAT TTC AGA CTC CTT GGC CCC AAT CGG G< 11580             11530        11540         11550        11560        11570 AA CTG GAT CAG ATC TCT GGA ATT GGT CCG AAA CCA AGT TCG TCT AAA TCC ATT CAA TGA G< 11640             11590        11600         11610        11620        11630 AT CTT GTT CAA TCA GCT ATG TCG TAC AGT GGA TAA TCA TTT GAA ATG GTC AAA TTT GCG A< 11700             11650        11660         11670        11680        11690 AA AAA CAC AGG AAT GAT TGA ATG GAT CAA TAG ACG AAT TTC AAA AGA AGA CCG GTC TAT A< 11760             11710        11720         11730        11740        11750 CT GAT GTT GAA GAG TGA CCT ACA CGA GGA AAA CTC TTG GAG AGA TTA AAA AAT CAT GAG G< 11820              11770        11780         11790        11800                                                  >VSV Trailer                                                 |AG ACT CCA AAC TTT AAG TAT GAA AAA AAC TTT GAT CCT TAA GAC CCT CTT GTG GTT TTT A< 11880             11830        11840         11850        11860        11870 TT TTT TAT CTG GTT TTG TGG TCT TCG Tgg ccg gca tgg tcc cag cct cct cgc tgg cgc c< 11940             11890        11900         11910        11920        11930                >Hepatitis Delta Virus Ribozyme                |gg ctg ggc aac att ccg agg gga ccg tcc cct cgg taa tgg cga atg gga cct gct aac a< 12000             11950        11960         11970        11980        11990 aa gcc cga aag gaa gct gag ttg gct gct gcc acc gct gag caa taa cta gca taa ccc c< 12060             12010        12020         12030        12040        12050 tt ggg ggc tct aaa cgg gtc ttg agg ggt ttt ttg ctg aaa gga gga act ata tcc gga t< 12120             12070        12080         12090        12100        12110        >T7 Terminators        |gc ggc cga tcc ggc tgc taa caa agc ccg aaa gga agc tga gtt ggc tgc tgc cac cgc t< 12180             12130        12140         12150        12160        12170 ga gca ata act agc ata acc cct tgg ggc ctc taa acg ggt ctt gag ggg ttt ttt gct g< 12240             12190        12200         12210        12220        12230 aa agg agg aac tat atc cgg gtt aac ctg cat taa tga atc ggc caa cgc gcg ggg aga g< 12300             12250        12260         12270        12280        12290 gc ggt ttg cgt att ggg cgc tct tcc gct tcc tcg ctc act gac tcg ctg cgc tcg gtc g< 12360             12310        12320         12330        12340        12350 tt cgg ctg cgg cga ggg gta tca gct cac tca aag gcg gta ata cgg tta tcc aca gaa t< 12420             12370        12380         12390        12400        12410 ca ggg gat aac gca gga aag aac atg tga gca aaa ggc cag caa aag gcc agg aac cgt a< 12480             12430        12440         12450        12460        12470 aa aag gcc gcg ttg ctg gcg ttt ttc cat agg ctc cgc ccc cgt gac gag cat cac aaa a< 12540             12490        12500         12510        12520        12530 at cga cgc tca agt cag agg tgg cga aac ccg aca gga cta taa aga tac cag gcg ttt c< 12600             12550        12560         12570        12550        12590 cc cct gga agc tcc ctc gtg cgc tct cct gtt ccg acc ctg ccg ctt acc gga tac ctg t< 12660             12610        12620         12630        12640        12650 cc gcc ttt ctc cct tcg gga agc gtg gcg ctt tct caa tgc tca cgc tgt agg tat ctc a< 12720             12670        12680         12690        12700        12710 gt tcg gtg tag gtc gtt cgc tcc aag ctg ggc tgt gtg cac gaa ccc ccc gtt cag ccc g< 12780             12730        12740         12750        12760        12770 ac cgc tgc gcc tta tcc ggt aac tat cgt ctt gag tcc aac ccg gta aga cac gac tta t< 12840             12790        12800         12810        12820        12830 cg cca ctg gca gca gcc act ggt aac agg att agc aga gcg agg tat gta ggc ggt gct a< 12900             12850        12860         12870        12880        12890 ca gag ttc ttg aag tgg tgg cct aac tac ggc tac act aga agg aca gta ttt ggt atc t< 12960             12910        12920         12930        12940        12950 gc gct ctg ctg aag cca gtt acc ttc gga aaa aga gtt ggt agc tct tga tcc ggc aaa c< 13020             12970        12990         12990        13000        13010 aa acc acc gct ggt agc ggt ggt ttt ttt gtt tgc aag cag cag att acg cgc aga aaa a< 13080             13030        13040         13050        13060        13070 aa gga tct caa gaa gat cct ttg atc ttt tct acg ggg tct gac gct cag tgg aac gaa a< 13140             13090        13100         13110        13120        13130 ac tca cgt taa ggg att ttg gtc atg aga tta tca aaa agg atc ttc acc tag atc ctt t< 13200             13150        13160         13170        13180        13190                                                                              >psP72                                                                             | ta aat taa aaa tga agt ttt aaa tca atc taa agt ata tat gag taa act tgg tct gac a< 13260             13210        13220         13230        13240        13250 gt tac caa tgc tta atc agt gag gca cct atc tca gcg atc tgt cta ttt cgt tca tcc a< 13320             13270        13280         13290        13300        13310 ta gtt gcc tga ctc ccc gtc gtg tag ata act acg ata cgg gag ggc tta cca tct ggc c< 13380             13330        13340         13350        13360        13370 cc agt gct gca atg ata ccg cga gac cca cgc tca ccg gct cca gat tta tca gca ata a< 13440             13390        13400         13410        13420        13430 ac cag cca gcc gga agg gcc gag cgc aga agt ggt cct gca act tta tcc gcc tcc atc c< 13500             13450        13460         13470        13480        13490 ag tct att aat tgt tgc cgg gaa gct aga gta agt agt tcg cca gtt aat agt ttg cgc a< 13560             13510        13520         13530        13540        13550 ac gtt gtt gcc att gct aca ggc atc gtg gtg tca cgc tcg tcg ttt ggt atg gct tca t< 13620             13570        13590         13590        13600        13610 tc agc tcc ggt tcc caa cga tca agg cga gtt aca tga tcc ccc atg ttg tgc aaa aaa g< 13680             13630        13640         13650        13660        13670 cg gtt agc tcc ttc ggt cct ccg atc gtt gtc aga agt aag ttg gcc gca gtg tta tca c< 13740             13690        13700         13710        13720        13730 tc atg gtt atg gca gca ctg cat aat tct ctt act gtc atg cca tcc gta aga tgc ttt t< 13800             13750        13760         13770        13780        13790 ct gtg act ggt gag tac tca acc aag tca ttc tga gaa tag tgt atg cgg cga ccg agt t< 13860             13810        13820         13830        13840        13850 gc tct tgc ccg gcg tca ata cgg gat aat acc gcg cca cat agc aga act tta aaa gtg c< 13920             13870        13880         13890        13900        13910 tc atc att gga aaa cgt tct tcg ggg cga aaa ctc tca agg atc tta ccg ctg ttg aga t< 13980             13930        13940         13950        13960        13970 cc agt tcg atg taa ccc act cgt gca ccc aac tga tct tca gca tct ttt act ttc acc a< 14040             13990        14000         14010        14020        14030 gc gtt tct ggg tga gca aaa aca gga agg caa aat gcc gca aaa aag gga ata agg gcg a< 14100             14050        14060         14070        14080        14090 ca cgg aaa tgt tga ata ctc ata ctc ttc ctt ttt caa tat tat tga agc att tat cag g< 14160             14110        14120         14130        14140        14150 gt tat tgt ctc atg agc gga tac ata ttt gaa tgt att tag aaa aat aaa caa ata ggg g< 14220             14170        14180         14190        14200        14210 tt ccg cgc aca ttt ccc cga aaa gtg cca cct gac gtc < 14258            14230        14240         14250 

Features: T7-g10 Promoter: [1:49] Hammerhead Ribozyme: [50:107] VSVLeader: [108:170] N: [171:1439] P: [1503:3049] M: [2360:3049]

Env.BG505 immunogen: [3198:5357]

L: [5479:11808] VSV Trailer: [11809:11907] Hepatitis Delta VirusRibozyme: [11913:11991] T7 Terminators: [11992:12260]

pSP72: [12261:14258]

SEQ ID NO: 2: 1mkcllylafl figvnckasa enlwvtvyyg vpvwkdaett lfcasdakay etekhnvwat 61hacvptdpnp qeihlenvte efnmwknnmv eqmhtdiisl wdqslkpcvk ltplcvtlqc 121tnvtnnitdd mrgelkncsf nmttelrdkk qkvyslfyrl dvvqinenqg nrsnnsnkey 181rlincntsai tqacpkvsfe pipihycapa gfailkckdk kfngtgpcps vstvqcthgi 241kpvvstqlll ngslaeeevm irsenitnna knilvqfntp vqinctrpnn ntrksirigp 301gqafyatgdi igdirqahct vskatwnetl gkvvkqlrkh fgnntiirfa nssggdlevt 361thsfncggef fycntsglfn stwisntsvq gsnstgsnds itlpcrikqi inmwqrigqa 421myappiqgvi rcvsnitgli ltrdggstns ttetfrpggg dmrdnwrsel ykykvvkiep 481lgvaptrakr rvvgrekrav gigavflgfl gaagstmgaa smtltvqarn llsgivqqqs 541nllraieaqq hllkltvwgi kqlqarvlav erylrdqqll giwgcsgkli cttnvpwnss 601wsnrnlseiw dnmtwlqwdk eisnytqiiy glleesqnqq ekneqdllal dkwaslwnwf 661disnwlwyik ssiasfffii gliiglflvl rvgiylcikl khtkkrqiyt diemnrlgkVSV G signal peptide Ala-Ser amino acid linker Env.BG505 ectodomainVSV G transmembrane region VSV G cytoplasmic tail

It is to be understood and expected that variations in the principles ofinvention as described above may be made by one skilled in the art andit is intended that such modifications, changes, and substitutions areto be included within the scope of the present invention.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

Example 1: VSVΔG-Env.BG505 Vaccine—Live Attenuated VSV-HIV ChimeraDelivering Env Trimers

Vesicular stomatitis virus (VSV) has been modified to generate a livechimeric virus vaccine (VSVΔG-Env.BG505) for active immunization againstHIV. The replication-competent recombinant chimera delivers a functionalHIV Env glycoprotein trimer (clade A.BG505) in the context of viralreplication mimicking native HIV spike presentation during an HIVinfection.

The VSVΔG-Env.BG505 chimera was constructed by replacing the natural VSVglycoprotein (G) gene with coding sequence for Env.BG505 (FIG. 1). As aresult, Env is the only transmembrane glycoprotein encoded by thechimera, and virus propagation and spread is dependent on expression offunctional Env trimers and infection of CD4+/CCR5+ cells.

VSVΔG-Env.BG505 is generated from a VSV genomic DNA clone that wasdeveloped from a lab-adapted strain of VSV (Indiana serotype). Thegenomic sequence is similar, but not identical, to the VSV genomic clonedeveloped at Yale University (1), which is used for other VSV-basedvaccine candidates including the attenuated VSV-N4CT1 vector developedby Profectus and NIAID (2), and the VSVΔG-Ebola virus chimera developedby the National Microbiology Laboratory in Canada (3), NewLink Genetics,and Merck Vaccines (4-6). About 100 nucleotides out 11 kb differ betweenthe Yale and IAVI genomic clones.

Live VSVΔG-Env.BG505 is recovered from plasmid DNA by electroporatingcells with the modified VSV genomic clone (FIG. 1C), a plasmid encodingT7 RNA polymerase to synthesize genomic RNA copies, and five plasmidsthat provide VSV polypeptides (N, P, M, G, L) in trans to initiate virusreplication (9). The virus rescue protocol does not require proprietarytransfection reagents or helper virus, and it has been optimized for usewith Vero cell substrates (protocol adapted from (10, 11)). Recovery ofinfectious VSVΔG-Env.BG505 can be initiated by electroporating plasmidsinto Vero cells derived from a qualified cell bank (cells from MeridianLife Science, Inc. are used at IAVI), after which the virus must bepropagated in cells that express CD4 and CCR5 to support Env-dependentreplication. Thus, recombinant virus amplification, clonal isolation,virus seed preparation, and vaccine production is performed with amodified Vero cell line that contains the genes for human CD4 and CCR5(VeroCD4/CCR5).

Applicants developed a stable VeroCD4/CCR5 cell line for propagation ofthe VSVΔG-Env.BG505 chimera. The cell line used in the lab currentlyencodes human CD4 and CCR5 and was developed under research conditionsstarting with cells obtained from the Meridian Life Science qualifiedVero cell bank. The research VeroCD4/CCR5 cell line is stable and hasbeen used for several years to support work on VSVΔG-Env.BG505 and anumber of similar chimeric viruses. Yields of VSVΔG-Env.BG505 producedin VeroCD4/CCR5 monolayers typically are >1×10⁷ pfus per ml of harvestedculture medium. Work on deriving a new cell line has been initiated forthe purpose of generating VeroCD4/CCR5 cells that will meet requirementsassociated with future VSVΔG-Env.BG505 cGMP manufacturing. As usedherein, VERO-CD4/CCR5, VeroCD4/CCR5 and VERT or VERT3 are usedinterchangeably.

The VSVΔG-Env.BG505 vaccine tested in rhesus macaques contained‘pseudotyped’ (12) virus particles to enhance vaccine uptake and promotea vigorous initial round of infection and replication. When virus wasgrown to produce a batch of vaccine, infection was conducted underconditions in which the VSV G glycoprotein was transiently expressed inVeroCD4/CCR5 cells allowing production of particles containing G. Anefficient laboratory method was developed to simplify addition of the Gpseudotype. A suspension of VeroCD4/CCR5 cells is mixed with plasmid DNAencoding G and VSVΔG-Env.BG505 particles after which the mixture issubjected to electroporation. The electroporated cells are thendistributed into cell factories containing culture medium. Virus isharvested and purified 48 hours post-electroporation.

Two points about G pseudotyping and the VSVΔG-Env.BG505 vaccine areworth emphasizing. First, VSVΔG-Env.BG505 does not contain the G gene;thus, infected cells do not express G and the VSV glycoprotein ispresent only in pseudotyped virus particles used for vaccination.Following vaccination, progeny VSVΔG-Env.BG505 particles produced by thefirst round of replication will lack the G glycoprotein making allsubsequent rounds of infection dependent on HIV Env and infection ofCD4+/CCR5+ cells of lymphoid origin. Because G is present onlytransiently (FIG. 2), it cannot promote spread of infection to othertypes of cells and tissues (i.e. neurons in the central nervous system).

The second point is related to the benefit of the pseudotyping. It iswell established that G is a very effective virus attachment protein,which has been used to pseudotype a variety of different candidate viralvaccines, gene therapy vectors, and oncolytic agents (12-15). A positiveeffect of pseudotyping on immunogenicity of a prototype VSVΔG-SIV Envchimera was demonstrated experimentally in a small pilot macaque studyin which animals were vaccinated mucosally (combination of oral andnasal cavity) with a vaccine prepared with and without a G pseudotype.In animals vaccinated with pseudotyped virus particles, anti-SIV Envantibody titers were greater than 100-fold higher (FIG. 5). Moreover,the transient exposure to G in the virus inoculumn did not elicitsignificant titers of anti-G antibodies (data not shown).

Many different glycoproteins may be used to pseudotype VSV particlesbesides G. Alternative pseudotypes may be useful for targeting vaccinedelivery to different areas. Examples include F plus H frommorbilliviruses, the F and HN from various parainfluenza viruses, the Fand G from various pneumoviruses, the F plus HN from various rubullaviruses. Also, the glycorpteins from filoviruses or arena viruses, amongothers.

Although the efficacious VSVΔG-Env.BG505 vaccine was a pseudotypedparticle, it is important to note that studies have not yet been done inmacaques to assess whether pseudotyping contributes to vaccine efficacy.Furthermore, G was selected for pseudotyping because it was known to behighly effective, but other alternative viral glycoproteins can be usedif it becomes necessary to develop a pseudotyped vaccine that targets amore limited cell population.

The VSVΔG-Env.BG505 vaccine was designed to deliver authentic HIVenvelope (Env) trimers mimicking the presentation of Env spikes by HIVinfection or a live attenuated HIV vaccine. As designed, the replicationcompetent chimeric virus provides several important immunostimulantsonce administered, including: 1) innate signaling initiated by infectionand replication of an RNA virus; 2) infected cells containing Envincorporated in the cell surface membrane; and 3) progeny virusparticles containing Env spikes arrayed on their surface. Moreover, likeHIV or SIV, Env-dependent VSVΔG-Env.BG505 propagation in vivo mightcontribute to vaccine efficacy by providing more persistent antigenexposure and immune stimulation that is associated with infectionoccurring in lymphoid tissues (16, 17).

VSVΔG-Env.BG505 is designed to propagate using Env as its attachment andentry protein. This has several important consequences during chimericvirus replication in the vaccinee, including: 1) there is strongselective pressure to maintain the gene encoding functional Env; 2) itensures that the replicating chimeric virus will present the immunesystem with authentic Env spike targets; and 3) because Env isfunctional and incorporated in the membrane, it has the conformationalflexibility of a native spike and will expose the immune system with afull range of authentic antigenic determinants. Related to the lastpoint, it also is important to emphasize that the functional Env.BG505trimer expressed by VSVΔG-Env.BG505 is not a conformationallyconstrained trimer like some other experimental vaccines that have beendevelop recently like Env.BG505 SOSIP or Env.BG505 NFL described byothers (18, 19).

Rose and colleagues first demonstrated that it was feasible to generatean infectious VSVΔG-Env chimera using a clade B Env (23), but additionaldevelopment was necessary to advance an effective vaccine candidate.First, the Env.BG505 immunogen was selected specifically because it wasknown to have a broad antigenicity profile (24) and it was isolated froman infected infant that produced bnAbs (25, 26). Second, it wasnecessary to investigate Env modifications for a number of reasons,including 1) to ensure Env gene genetic stability; 2) to enable vigorousreplication in cell culture that would support vaccine production; and3) to substantially increase Env incorporation into to the infected cellmembrane and virus particle to provide improved display of Env spikeimmunogens. Following an approached suggested by earlier data showingthat the Env cytoplasmic tail caused vector genetic instability(unpublished and (27)) and suppressed incorporation into VSV particles(28), a number of hybrid Envs were designed and evaluated (FIG. 3) inwhich various combinations of the Env signal peptide (SP), transmembrane(TM) region, and cytoplasmic tail (CT) were replaced with sequence fromVSV G (Indiana serotype). A hybrid Env containing the VSV G SP, TM andCT was found to be expressed abundantly on the cell surface oftransfected cells and also was found to support efficient Env-dependentreplication of the VSVΔG-Env.BG505 chimera in CD4+/CCR5+ cells. A hybridin which the Env membrane-proximal external region (MPER) also wasreplaced with the analogous ‘Stem’ domain of G was expressed in modestlygreater quantities on the surface of transfected cells, but since itlacked the important Env MPER epitope, all subsequent vector design hasfocused on the Env hybrids where the SP, TM, and CT are substituted withVSV G sequences. Therefore, the VSVΔG-Env.BG505 vaccine encodes a highlyexpressed Env-G hybrid, which is designed so that all sequence displayedon the membrane surface is Env ectodomain while intracellular andmembrane-spanning sequences are derived from G.

The Env-G hybrid immunogen incorporated on the surface of infected cellsand VSVΔG-Env.BG505 particles is broadly antigenic. An example ofinfected cells analyzed by flow cytometry (FIG. 4B) shows that multiplemAbs bind the cell surface including PGT145, PGT151 and VRC06, whichbind preferentially to determinants formed by well-ordered trimmers (19,21, 22). Similarly, bnAbs recognize purified virions when they areadsorbed to alum and the alum-virus complexes are analyzed by flowcytometry (FIG. 4D), which agrees with electron microscope images (FIG.4C) showing surface density consistent with the present of glycoproteincomplexes on the surface of VSVΔG-Env.BG505 particles.

Part of the vaccine design objective was to develop a chimeric virusthat could be administered effectively by a mucosal route to stimulateimmune protection at the mucosal barrier. Even though a mucosalapplication of the live vaccine may be advantageous, Applicants do notenvision the vaccine to be limited to this route of administration.Because research and development on lentivirus virus vectors has shownthat Env is not an effective attachment protein for virus particledelivery, VSVΔG-Env.BG505 modifications were considered that mightsignificantly improve virus uptake without changing the key feature ofthe chimeric virus, which is its unique design in which Env is the soleglycoprotein expressed following infection. Thus, rather thangenetically modifying the VSVΔG-Env.BG505 vector further, a decision wasmade to test vaccines in which the virus particles were prepared with aG pseudotype, as a considerable body of work on lentiviruses (12) aswell as a variety of chimeric VSV vectors (29) showed that pseudotypingwith G was effective.

To support testing of a pseudotyped VSVΔG-Env vaccine, a simple systemwas developed to add G to virus particles. Briefly, a suspension ofVeroCD4/CCR5 cells is mixed with plasmid DNA encoding G andVSVΔG-Env.BG505 particles and then mixture is subjected toelectroporation. The electroporated cell suspension is then distributedinto cell stacks and cultured for ˜48 hours after which pseudotypedvirus particles are harvested and purified. The efficiency ofpseudotyping can then be quantified by evaluating plaque formation onCD4+/CCR5+ cell monolayers in which Env or G can direct infection, andcomparing this to G-mediated infection of standard Vero monolayers,which support a single-cycle of infection that can be quantified byimmunostaining to detect individual cells expressing viral proteins.

A pilot study was conducted in Indian rhesus macaques with a prototypeVSVΔG-Sly chimera (FIG. 5A, VSVΔG-SIV-GagEnv). Macaques were used forthis early study because transgenic or ‘humanized’ small animal modelsthat can support replication of a CD4/CCR5-tropic virus havelimitations. The macaque study was conducted for three primaryreasons: 1) assess the ability to safely vaccinate mucosally in thenasal and oral cavity with a chimeric virus; 2) detect and quantifyserum anti-Env antibodies elicited by mucosal vaccination; and 3)compare vaccines prepared with and without a G pseudotype.

Macaques were vaccinated (FIG. 5B) at weeks 0 and 6 by applying virussolution to the nasal and oral cavities (1×10⁸ pfus per site).Importantly, animal behavior was normal following vaccination and nolesions were observed in or around the nose or mouth. Quantification ofantibody titers by bioplex assay (30) showed that the chimeric virusvaccines were immunogenic and that the pseudotyped vaccine wassignificantly more potent. Following the first vaccination, samplesanalyzed at week 6 showed that the pseudotyped VSVΔG-SIV-GagEnv vaccineelicited low but detectable antibody titers, while animals vaccinatedwith an ‘empty’ VSV vector or the chimeric virus lacking the Gpseudotype had values near baseline. Env antibody titers increased afterhomologous boost at week 6, and it was clear that the peak titerelicited by the pseudotyped chimera was considerably stronger (>1,000×)compared to the magnitude of the response generated by the vaccinelacking the G pseudotype, and the titers also remained substantial >2.5months after the week-6 boost. It also is worth highlighting that twomucosal vaccinations with the pseudotyped VSVΔG-SIV-GagEnv vaccinegenerated antibody titers that were in the same range as peak responsesseen with a relatively potent vaccination regimen based on 3×DNA-SIV-Envprime (intramuscular electroporation) and Ad5-SIV Env (intramuscular)boost (31).

Several conclusions were drawn from this pilot study. First, thechimeric virus vaccine was able to safely elicit anti-Env antibodiesagainst a membrane anchored Env spike. Second, antibody titers of thismagnitude elicited by mucosal vaccination indicated that the VSVΔG-SIVchimera replicated following vaccination and that the antibody responsewas not elicited simply by exposure to the virus particles delivered ina buffered solution. This assumption also is consistent with the factthat G in the pseudotyped particles did not elicit an anti-G responsesignificantly above background in an ELISA (data not shown). Finally, itwas evident that the chimeric virus vaccine prepared with the Gpseudotype was more immunogenic, thus the HIV vaccine based onVSVΔG-Env.BG505 was advanced for testing in macaques as a pseudotypedvaccine.

The preclinical efficacy of the VSVΔG-Env.BG505 vaccine prepared with aG pseudotype is being evaluated in Indian rhesus macaques using therectal SHIV challenge model. The study was designed with the three mainobjectives: 1) show that the VSVΔG-Env.BG505 chimera could beadministered safely to the nasal and oral cavities; 2) demonstrate thatvaccination elicits anti-Env antibodies; and 3) establish thatvaccination provides measurable protection from rectal exposure with aheterologous clade B SHIV (SHIV SF162p3).

The study also included a head-to-head comparison with a second VSVvector encoding the same Env.BG505 trimer immunogen. The main purpose ofthis comparison was to evaluate an alternative Env.BG505 delivery vector(VSV-G6-Env.BG505, FIG. 6) that would have increased replicativecapacity in vivo. To achieve greater replicative capacity, theVSV-G6-Env.BG505 vector was designed to contain genes encoding Env.BG505and G; therefore, the vector coexpresses the glycoproteins in infectedcells, and incorporate both trimeric complexes in virus particles. Asdesigned, the VSV-G6-Env.BG505 vector can propagate and spread in awider range of cells in vivo because the continuous expression of Gallows infection and spread into a much broader range of cell types.Thus, both the pseudotyped VSVΔG-Env.BG505 chimera and VSV-G6-Env.BG505can infect most cell types at the site of vaccine administration usingG, but after the initial round of replication, secondary infectioninitiated by progeny virus particles will be significantly different,with the VSVΔG-Env.BG505 targeted specifically to CD4+/CCR5+ cells andVSV-G6-Env.BG505 being able to spread into multiple cell types.

The preclinical efficacy study was designed with three groups of 10macaques (negative for Mamu-B*08 and -B*17 MHC alleles associated withimmune control) that were vaccinated at weeks 0, 4, and 29 withpseudotyped VSVΔG-Env.BG505, VSV-G6-Env.BG505 or a saline control. It isimportant to highlight that vaccination was conducted only with the liveVSV vectors, and no boost was performed with a heterologous vaccine.Vaccines were administered by application to mucosal surfaces in thenasal and oral cavity of sedated animals (1×10⁸ pfus per site). No locallesions were observed and all macaques behaved normally aftervaccination.

All macaques immunized with a VSV-based Env.BG505 vaccine developeddetectable anti-Env serum antibodies after the second vaccination. Thethird vaccination at week 29 provided a boost, and perhaps moreimportantly, increased the durability of the antibody titiers, whichpersisted during the 5-month rest period before challenge in 8 out of 10macaques vaccinated with VSVΔG-Env.BG505 and all animals vaccinated withVSV-G6-Env.BG505. The TZM-bl assay (33) also was used to analyse serumfor virus-neutralizing antibodies (nAbs). The resulted showed that nAbtiters were low (titers ≤100) and were detectable in only somevaccinated animals (summarized on the ELISA chart in FIG. 8). Inmacaques vaccinated with the VSVΔG-Env.BG505 chimera, 4 animals werepositive for nAbs active against HIV SF162p3 pseudovirus at week 31, butthe titers waned to undetectable by the day of SHIV challenge.Vaccination with VSV-G6-Env.BG505 elicited nAbs against SF162p3 andhomologous BG505 pseudovirus that were detectable at week 31 and 48, butnot in all animals.

Clade B SHIV SF162p3 challenge commenced at week 48, which was about 5months after the final vaccination (FIG. 7). The challenge protocol wascomposed of 3 stages: the first 5 rectal exposures conductedapproximately every two weeks, a 6-week rest period, and the final 5biweekly exposures. Macaques with ≥200 genome SHIV copies per ml ofplasma were considered infected after which challenge was stopped. Allinfected macaques were viremic for weeks following the initial infection(FIG. 9) as determined by detection of SHIV genomes in the blood, andaccordingly, the infected animals developed antibodies against Gagexpressed by the SHIV (data not shown).

The SHIV infection rate was significantly reduced in macaques vaccinatedwith the VSVΔG-Env.BG505 chimera compared to animals vaccinated withVSV-G6-Env.BG505 or saline control (FIG. 10). Over the course of 9challenges, 9 out of 10 macaques in the Control and VSV-G6-Env.BG505groups became chronically infected with SHIV at a similar frequency. Incontrast, in macaques vaccinated with VSVΔG-Env.BG505, just 3 wereinfected with challenge virus indicating that VSΔG-Env.BG505immunization significantly increased resistance to mucosal SHIVinfection. Thus, vaccine efficacy as measured by prevention of rectalinfection with a heterologous clade B SHIV was 67%.

Immunologic assessment continues, but current results point to apotential relationship between the reduced frequency of infection seenin the animals vaccinated with the VSVΔG-Env.BG505 vaccine (FIG. 10) andEnv-specific serum antibodies. First, both replication-competent VSVvectors elicited serum antibodies that persisted for the 5-month periodbetween the final vaccination and the beginning of the repetitive SHIVchallenge protocol (FIG. 8). In the animals vaccinated withVSVΔG-Env.BG505, there were 2 animals in which the antibody titers wanedto baseline levels by week 48 when challenge commenced (FIG. 8), andinterestingly, these were the same two animals that appeared leastresistant in this group and became infected by challenge 2 (FIG. 10).The third animal that became infected in this group resisted 7challenges conducted over a period of about 4.5 months, but becameinfected at exposure 8 by which time the serum antibody titers hadwaned. These results imply that there is a relationship betweenEnv.BG505 gp120 binding antibody titers and SHIV infection resistance.This trend is summarized graphically in FIG. 11.

In contrast to the results seen in animals vaccinated with theVSVΔG-Env.BG505 chimera, that rate of infection in macaques vaccinatedwith VSV-G6-Env.BG505 was very similar to the control group indicatingthat vaccination did not have measurable effect on SHIV infectionfrequency (FIG. 10). This was observed even though all animals haddeveloped Env antibodies in response to vaccination, including somemacaques that had nAbs (FIG. 8).

Taken together, the results of vaccination with the different liveVSV-based vectors show that both types of vaccine elicit Env antibodies,but that the quality of the antiviral immunity is very different. Whatis responsible for this difference in protection is not understood atthis time, but perhaps it is related to antibody binding sitespecificity, the diversity of Env epitopes recognized, or IgG effectorfunctions. Alternatively, the two live vectors might elicit differentprofiles of Env-specific T cells with antiviral activity that isaffecting infection resistance. Ongoing and future immunologicassessment will help identify differences in the immune responseselicited by the two vaccines, which will provide guidance for vector andimmunogen improvements.

The results produced with two different replicating VSV-based vaccinesalso illustrates clearly that specific vaccine design details can have apronounced effect on efficacy. Some of the unique features of theVSVΔG-Env.BG505 vaccine that might contribute to efficacy, include; 1)CD4+/CCR5+ tropism that targets replication to lymphoid cells andtissues; 2) chimeric virus propagation in vivo that is dependent onexpression of functional Env and will provide immune system exposure toauthentic Env spikes; 3) the only glycoprotein expressed is Env, thusthere is no other competing glycoprotein immunogen that might dominateimmune responses; and 4) the lack of other vector-encoded glycoproteinseliminates development of potent anti-vector antibodies that mightinterfere with multiple immunizations.

Preclinical efficacy in the SHIV challenge model was observed followingmucosal vaccination with a total dose of 2×10⁸ pfu per ml. The vaccinedose was split between two sites. Mucosal surfaces in the nasal and oralcavities each received 1×10⁸ pfu applied in a buffered solution.

Preclinical efficacy in the SHIV challenge model was observed with avaccination schedule of 0, 4, and 29 weeks. Other vaccination schedulesare also contemplated.

Mucosal vaccination in the nasal and oral cavity was tested primarilybecause the goal was to stimulate enhanced mucosal immunity. Otherconsiderations supporting this vaccination route included: 1) providingaccess to submucosal CD4+/CCR5+ lymphocytes that would be targets forVSVΔG-Env.BG505 replication, and 2) VSV naturally infects these mucosalsites.

VSVΔG-Env.BG505 is a recombinant chimeric virus based on the VSV Indianaserotype. The VSV G gene deleted and replaced with sequence encodingfunctional HIV Env.BG505. The live vaccine is replication competent andpropagates specifically in cells that contain the CD4/CCR5 receptors.

The efficacious preclinical vaccine is a G-pseudotyped VSVΔG-Env.BG505that is applied to nasal and oral cavity mucosal surfaces at 0, 4, and29 weeks.

The VSVΔG-Env.BG505 vaccine is expected to be safe for use in humans,because 1) no observable adverse reactions occurred in vaccinatedmacaques, and 2) the chimeric virus design makes propagation dependenton Env, thus virus spread in vivo is restricted to lymphoid cell andtissues that express CD4 and CCR5 and will prevent virus replication inother sites like the central nervous system.

The preferred cell line for vaccine production is VeroCD4/CCR5, whichhas been used to support preclinical development of the VSVΔG-Env.BG505chimeric virus vaccine. A similar cell line must be ‘rederived’ usingconditions and materials that are consistent with using the cells forvaccine manufacturing. Manufacturing processes and steps are set forthin FIG. 12. Preparation of VSVΔG-Env.BG505 with G pseudotype is setforth in FIG. 13.

Preclinical material tested in macaques may be purified by 2 round ofcentrifugation through sucrose cushion and the method is based ontangential flow filtration.

Preclinical vaccine material is stored frozen (−80) in Hank's BalancedSalt Solution (HBSS) supplemented with 15% trehalose.

Raw material and biological starting material suitability, quality, andcharacterization (e.g., passage history of cell substrate and viral seedmaterial) may include:

-   -   Recombinant VSVΔG-Env.BG505 is generated from a plasmid DNA        containing a modified VSV genomic clone in which the G gene is        replaced with sequence encoding HIV Env.BG505. Rescue of        recombinant virus is initiated by electroporating the genomic        clone with supporting plasmids that direct expression of VSV N,        P, M, G, and L proteins and T7 phage RNA polymerase.    -   The VSV genomic clone is based on the VSV Indiana serotype.    -   The VeroCD4/CCR5 cell line used for preclinical development was        generated starting with Vero cells from a cell bank qualified        cell bank (obtained from Meridian Life Science, Inc). The        VeroCD4/CCR5 cell line was generated by microporating cells with        a plasmid that contains genes for expression of human CD4 and        CCR5 and the Neo resistance marker.    -   VeroCD4/CCR5 is typically propagated in monolayer cultures. Cell        factories are used for virus production. The cells are grown in        DMEM supplemented with 10% fetal bovine serum from certified        suppliers. Virus amplification can be conducted in monolayers in        which the medium is exchanged with serum-free growth medium such        as VPSFM.

Preclinical vaccine characterization may include:

-   -   Potency: Virus is quantified by plaque assay on VeroCD4/CCR5        cells. To confirm virus particles are pseudotyped with G,        standard Vero monolayers are infected and the single-cycle        infection in incubated overnight. Monolayers are subsequently        immunostained to quantify infected cells.

Additional vaccine virus characterization:

-   -   Genome copies (qPCR) per infectious unit    -   Genomic nucleotide sequence    -   Env insert integrity by PCR    -   Env expression by Western blot    -   Env expression detected on infected cells by flow cytometry and        bnAbs    -   Virus purity by denaturing gel electrophoresis and silver stain    -   Mycoplasma testing by PCR    -   Endotoxin testing

Assay development required to support lot release or productcharacterization may include:

-   -   Potency—see above, plaque assay and genome-to-pfu ratio    -   Safety:        -   Env insert integrity by PCR        -   Lack of VSV G gene by PCR        -   Genomic sequence        -   Infection of Vero cells with pseudotyped virus and            subsequent blind passage to confirm lack of CPE indicating            that virus is CD4/CCR5-dependent as expected

Preparation of reagents to develop assays may include:

-   -   Primers and probes are available to assess genomic sequences,        quantify genome copies, and specifically detect the Env gene        insert.    -   Antibodies that can neutralize the pseudotyped VSVΔG-Env.BG505        chimera are required for adventitious agent testing. Antibodies        recognizing the G pseudotype block infection in eggs, mice, and        most cell lines provided they do not express primate CD4/CCR5.

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Example 2: Vaccination with a Live Vesicular Stomatitis Virus-HIV EnvChimera Prevents SHIV Infection

Seven of 10 Indian rhesus macaques vaccinated with a novelreplication-competent vesicular stomatitis virus vector designed withfunctional HIV Env substituting for the native VSV glycoprotein remaineduninfected after repeated rectal challenge with a heterologous clade BSHIV.

HIV is a challenging vaccine target because its functional envelopeglycoproteins (Envs) are highly glycosylated, sequence diverse, andassembled into a compact trimeric complex (the Env spike) that restrictsepitope access. Moreover, vaccines based on non-native forms of Env areeither ineffective or provide limited protection. Therefore, Applicantsdeveloped a novel spike delivery vaccine (VSVΔG-Env.BG505) fromvesicular stomatitis virus (VSV) by replacing the VSV glycoprotein (G)with functional clade A HIV Env. Rhesus macaques vaccinated with liveVSVΔG-Env.BG505 developed Env antibodies, and importantly, 7 of 10remained uninfected after repeated rectal challenge with heterologousclade B SHIV SF162p3. In contrast, a second more typical VSV vectorexpressing both Env and G induced Env antibodies but failed to protect,showing that the VSVΔG-Env.BG505 vector design was associated withpreclinical efficacy. Applicants' results indicate that the VSVΔGchimeric virus platform is an important developing vaccine technologyfor HIV Env glycoprotein delivery.

HIV is a challenging vaccine target because its functional envelopeglycoproteins (Envs) are highly glycosylated, sequence diverse, andassembled into a compact trimeric complex (the Env spike) that restrictsepitope access. Moreover, vaccines based on non-native forms of Env areeither ineffective or provide limited protection. Therefore, Applicantsdeveloped a novel spike delivery vaccine (VSVΔG-Env.BG505) fromvesicular stomatitis virus (VSV) by replacing the VSV glycoprotein (G)with functional clade A HIV Env. Rhesus macaques vaccinated with liveVSVΔG-Env.BG505 developed Env antibodies, and importantly, 7 of 10remained uninfected after repeated rectal challenge with heterologousclade B SHIV SF162p3. In contrast, a second more typical VSV vectorexpressing both Env and G induced Env antibodies but failed to protect,showing that the VSVΔG-Env.BG505 vector design was associated withpreclinical efficacy. Applicants' results indicate that the VSVΔGchimeric virus platform is an important developing vaccine technologyfor HIV Env glycoprotein delivery.

Replication-Competent VSV-HIV Env Vaccine Vectors.

The VSVΔG-Env.BG505 chimera was developed by replacing the VSV G genewith sequence encoding functional Env.BG505 (FIGS. 19A-B with moredetail in the Materials and Methods). In addition to expressing Env andreplicating with the cell tropism of HIV, the VSVΔG-Env.BG505 chimerahas several other features to highlight. First, its dependence onEnv.BG505 for propagation ensures that some functionally-configured Envis expressed during viral replication that will expose the immune systemto authentic Env spikes. Second, because the vector lacks the G gene,negative effects caused by G expression are avoided, such as the VSVglycoprotein dominating B cell responses or inducing potent anti-vectorimmunity. Finally, cells infected with VSVΔG-Env.BG505 produce progenyvirus particles containing Env arrayed on their surface, which isexpected to substantially enhance immunogen presentation to Blymphocytes (11).

To directly investigate whether the live VSVΔG-Env.BG505 chimera wasadvantageous for the reasons mentioned above, VSV-G6-Env.BG505 (FIG.19C) was developed as a comparator for use in the macaque studydescribed below. VSV-G6-Env.BG505 is a more typical VSV vector in whichthe Env.BG505 gene was added as an extra transcription while retainingG. The vector was generated by reintroducing the G gene at the terminusof the negative-sense RNA genome (FIGS. 19A and C; Gin genome position6), which maintained Env in the same genomic position relative to thepromoter (FIG. 19A) as in VSVΔG-Env.BG505 and modestly downregulated Gexpression (16). VSV-G6-Env.BG505 propagates efficiently using G, whichrecognizes a ubiquitous cellular receptor that enables infection of abroad range of cell types (17); thus, including VSV-G6-Env.BG505 in thevaccine study allowed us to ask whether this G-dependent vector mightdeliver the Env spike more effectively, perhaps because constitutive Gexpression confers increased replicative capacity in vivo, a differentcell and tissue tropism, or both.

Env.BG505 expression by the two different VSV vectors was compared byinfecting cultured cells and conducting flow cytometry using monoclonalantibodies that bind a variety of Env epitopes (4-6). When VERO cells ora stable VERO derivative (VERO-CD4/CCR5) expressing human CD4 and CCR5were exposed to VSVΔG-Env.BG505, only the CD4+/CCR5+ cells were infectedas shown by Env detected on the cell surface (FIG. 19D). The expandedtropism conferred by G allowed VSV-G6-Env.BG505 to infect both celltypes although the intensity of Env surface staining was reducedcompared to VERO-CD4/CCR5 cells infected with VSVΔG-Env.BG505. The moreintense cell surface staining produced by VSVΔG-Env.BG505 infection wasdue at least in part to increased Env expression, which was detectableby Western blot analysis (data not shown), but it also was possible thatG co-expression by cells infected with VSV-G6-Env.BG505 had a negativeeffect on Env incorporation into the cell plasma membrane. It also isimportant to note that the panel of monoclonal antibodies used for flowcytometry included some that recognize native Env spikes structures(PGT145, PGT151, and VRCO6b) as well as others (IgGb6 and F105) thatbind epitopes that are exposed when the Env subunits are not assembledinto a compact spike (18-20). Infected cells were bound by allantibodies included in the panel demonstrating that multiple forms ofEnv were expressed on the cell surface including well-ordered Envspikes, as is typical for an HIV infection (21).

Because Env spikes arrayed on progeny virions produced duringreplication in vivo were expected to be important immunogens (11), theantigenicity of purified virus particles was analyzed with a modifiedflow cytometry assay. In this assay, virus particles are adsorbed toaluminum phosphate (alum) to generate alum-virus complexes that can beincubated with monoclonal antibodies and are large enough to be analyzedwith a flow cytometer (16). Subsequent analysis with the same monoclonalantibody panel showed that substantially more Env was incorporated inthe VSVΔG-Env.BG505 chimera compared to VSV-G6-Env.BG505 (FIG. 19E andnote different Y axes), which also was confirmed by Western blotting(data not shown). The flow cytometry data also showed that theantigenicity of VSVΔG-Env.BG505 virions was similar to the infected cellsurface (FIG. 19D), including binding by VRCO6b, PGT145 and PGT151. Insummary, analysis of purified virions showed that both VSVΔG-Env.BG505and VSV-G6-Env.BG505 contained Env, but the immunogen was considerablymore abundant in the VSVΔG chimeric virus particle.

Vaccination and Preclinical Efficacy.

Three groups of 10 male Indian rhesus macaques were vaccinated byadministering live vector or saline control to both intranasal andintraoral surfaces at 0, 4 and 29 weeks (FIG. 20A). The five-month breakbetween the second and third vaccination was included to provide timefor germinal center reactions and B cell differentiation (22). Allimmunizations were conducted with a VSV vector, and no boostervaccinations were administered with a heterologous vector or subunitvaccine.

No adverse reactions were observed after vaccination. Virus sheddinginto the oral cavity was analyzed using qRT-PCR, which showed that viralgenomes were low to undetectable for VSVΔG-Env.BG505 but increased forVSV-G6-Env.BG505 particularly following the first vaccination (FIG. 25).This result implied that the replicative capacity of VSV-G6-Env.BG505was greater, but it might also be due to differences in cell and tissuetropism affecting shedding into the oral cavity. VSV genomes were notdetected in the blood (data not shown) in either group, which wasconsistent with lack of viremia detected in earlier studies (23).Interestingly, VSVΔG-ZEBOV did cause transient viremia in macaques (13)and clinical trial participants (14, 15), which might reflect an effectof cell tropism conferred by the Ebola virus glycoprotein.

Intrarectal SHIV SF162p3 challenge commenced at week 48, about 4.5months after the third vaccination (FIG. 20A). This rest period prior toSHIV challenge allowed waning of peak adaptive immune responses as wellas decay of innate immunity that might have been triggered by VSV. Amaximum of 10 sequential challenges were planned (FIG. 20A), with thefirst five being conducted approximately every 2 weeks after which abrief rest period was included to allow innate immune responses todecline if any were induced by repeated SHIV exposure (24, 25).Challenged animals that had 200 SHIV genome copies or more on twosuccessive blood draws were considered positive (FIG. 26), at which timechallenge was stopped. All vaccinated animals that tested positive forSHIV genomes also developed antibodies against Gag expressed by the SHIV(FIG. 27).

After completing repetitive SHIV challenge, 9 of 10 placebo controlanimals were infected but just 3 of 10 in the VSVΔG-Env.BG505 group(FIG. 20B). This indicated that the VSVΔG-Env.BG505 group wassignificantly protected with an overall efficacy of 67% (P=0.014). Theper-challenge vaccine efficacy for VSVΔG-Env.BG505 was estimated to be79.8% based on a Leaky vaccine model (26). In contrast, vaccination withVSV-G6-Env.BG505 had no protective effect (FIG. 20B, and Table 1).

Antibody titers in animals 11 and 15 were at the lower measureable limitwhen SHIV challenge was initiated at week 48 (FIGS. 20A and B), and bothmacaques were infected right away at challenge 1 and 2, respectively(FIG. 20B with more detail in FIG. 29). Animal 16 resisted 7 challengesconducted over a period of ˜5 months (FIGS. 20B and 29). By challenge 8(week 67) when infection occurred in animal 16, titers had declined tonear baseline (FIG. 29B). Thus, in the three unprotected macaques in theVSVΔG-Env.BG505 group, low binding antibody titers were associated withthe timing of SHIV infection.

To determine what regions of Env might be targeted in response toVSVΔG-Env.BG505 vaccination, additional mapping of serum antibodybinding specificity was performed with several assays. For conductingELISAs and Western blots, seven different regions of Env.BG505 (FIG.22A) were expressed as fusion proteins using human serum albumin (HSA)as the N-terminal fusion partner. Fusion to HSA enabled expression ofthe Env.BG505 fragments as secreted glycoproteins (31). For the ELISAresults shown in FIG. 22B, the purified recombinant proteins werecaptured on microtiter plates (capture ELISA) using their C-terminal Histag after which they were reacted with sera from week 48. Fourconclusions can be drawn from the capture ELISA data. First, thepredominant positive signal in both vaccine groups was against theHSA-V3C3 and HSA-gp41 (gp41 ectodomain only). These fusion proteins alsogenerated the most frequent and intense signals when used in a Westernblot assay (FIG. 32). Second, sera from the three unprotected macaquesin the VSVΔG-Env.BG505 group (11, 15, and 16) had lower HSA-V3C3 andHSA-gp41 values consistent with these animals being low responders, asobserved earlier with Western blots (FIGS. 21A-B). Finally, the HSA-gp41substrate allowed unambiguous detection of antibodies specific for gp41in vaccinated animals (FIGS. 22B and 32), which were not observed in theearlier Western blot assays (FIGS. 21A and 30). Lack of gp41 signals inthe prior Western blots likely was due to lower gp41 quantities beingpresent on the blot membrane, but perhaps conformation assumed by thedifferent gp41 substrates played some role.

Seven of 10 macaques vaccinated with the live Env-dependentVSVΔG-Env.BG505 chimera remained uninfected after repeated rectalchallenge with heterologous clade B SHIV SF162p3 (FIG. 20). Notably,this level of efficacy was produced with a three-dose regimen ofVSVΔG-Env.BG505, which differentiates this vaccine from some othersevaluated before where protection was observed after immunization withmultiple types of vaccine used either in combination or in aheterologous prime-boost regimen (8, 33, 34).

In the 7 protected macaques vaccinated with VSVΔG-Env.BG505, resistanceto SHIV infection was associated with persistent Env-specific serumantibodies, while in the three animals that became infected, poorvaccine take or waning antibody titers were a marker of susceptibility(FIGS. 21, 22 and 29). Perhaps the most visual evidence for this was thegp120 ELISA data (FIG. 29) and Western blot results (FIG. 21), whichclearly showed that the unprotected animals had reduced quantities ofEnv-specific serum antibodies. Further analysis of the sera from thisgroup identified statistically significant correlations between themagnitude of antibody binding activity and SHIV infection resistance(6D), but it remains to be determined if the more abundant Envantibodies are directly involved in the protective mechanism or whetherthey primarily are indicators of VSVΔG-Env.BG505 vaccine take. Thesuggestion that they contribute to the mechanism of protection mightgain support from the data showing that gp120 V3 and gp41 (FIG. 22) wereprominent targets of the antibody response induced by VSVΔG-Env.BG505.Antibody binding to V3 and gp41 has been linked to protection before,for example, reduced infection risk was correlated with anti-V3antibodies in the RV144 clinical trial (32) and anti-gp41 antibodieshave been associated with protection from progressive SIV infection inthe macaque model (35).

Functional activities associated with the protective antibodies remainto be identified. Even if undetectable quantities of neutralizing serumantibodies were present, their activity likely would not be adequate tomediate protection (36). Maybe mucosal vaccination with VSVΔG-Env.BG505resulted in neutralizing antibody being tissue associated or in mucosalsecretions rather than in circulation, although anti-gp120 antibodieswere not detected in oral or rectal swab samples (data not shown). Itseems more likely that protection was due to Env-specificimmunoglobulins that direct antibody-mediated effector functions, likethose induced by other Env vaccine candidates evaluated in recentpreclinical studies (33, 34) or the RV144 clinical trial (8). There isgrowing recognition that antibodies lacking classic in vitro virusneutralizing activity contribute substantially to protection from viralinfections, as illustrated by some recent studies on influenza virus(37, 38); thus, further investigation and comparison of effectorfunctions associated with IgG induced by protective VSVΔG-Env.BG505 ornonprotective VSV-G6-Env.BG505 will be informative.

The Western blot results indicated that binding activity persisted forat least a year in protected animals (FIG. 21D). This might resemblewhat is observed during vaccination with live attenuated viruses like inthe measles vaccine. Antibody titers established by measles vaccinationare considerably lower than those reached during natural infection, butthe attenuated virus replicates sufficiently to establish durableprotective antibodies (39). There likely is a similar requirement forVSVΔG-Env.BG505 to achieve replication threshold that provides anadequate quantity and duration of Env expression, results in release ofimmunogenic virus particles containing Env arrayed on their surface(11), and distributes immunogen to lymphoid tissues (40). Possibly,vaccine failure in the three unprotected macaques in the VSVΔG-Env.BG505group was caused by inadequate replication, thus future studies thatinvestigate VSVΔG-Env.BG505 propagation in vivo will be important.

Replicative capacity might also contribute to a notable differencebetween the VSVΔG-ZEBOV and VSVΔG-Env.BG505 chimeric virus vaccines. Inpreclinical and clinical studies (13-15), a single vaccination withVSVΔG-ZEBOV was sufficient for efficacy. A single vaccination withVSVΔG-ZEBOV may be sufficient because the virus apparently replicatesand disseminates more extensively based on finding virus in the blood ofmacaques and clinical trial volunteers (13-15). This suggests thatfurther development of the VSVΔG-Env.BG505 vaccine may benefit frominvestigating how to safely increase virus replication. This might beachieved by launching a more robust initial infection using a differentvaccination route or higher dose, or alternatively, maybe aVSVΔG-Env.BG505 vector can be developed that has increased replicativecapacity. A follow up study in macaques is being initiated toinvestigate some of these variables.

The VSVΔG chimeric virus design appears to be emerging as an importantvaccine technology for delivery of viral glycoprotein immunogens. TheVSVΔG-ZEBOV clinical trials showed that the Ebola virus vaccine was safeand efficacious (13-15). Promising preclinical results also have beenproduced with other hemorrhagic fever virus glycoproteins (41), and nowApplicants' data shows that this strategy can be adapted for use with anHIV Env trimer immunogen, which is well known to be a very difficultvaccine target (5). The effectiveness of the VSVΔG chimera designprobably is due to its ability to reproduce features of a naturalpathogen infection without pathology that inhibits development ofprotective adaptive immunity. Vaccine features such as expression of thenative transmembrane glycoprotein on the surface of infected cells,infection directed to cells and tissues specified by the tropism of theforeign glycoprotein, and the presentation of immunogen arrayed on virusparticles all likely play important roles in shaping the immuneresponse. Moreover, the lack of a G gene in the vector is veryimportant, because it eliminates expression of a dominant off-target Bcell immunogen, prevents development of potent anti-G antibodies, andallows the foreign glycoprotein to be repetitively arrayed on the virusparticle without interference from G.

To evaluate whether the promising preclinical performance ofVSVΔG-Env.BG505 can be extended to humans, as it was for the VSVΔG-ZEBOVvaccine, Applicants are developing a clinical trial candidate. It isrelevant to clinical development to mention that the G gene deletion inVSVΔG-ZEBOV resulted in loss of the VSV neurovirulence phenotype that isobserved in some preclinical models (42). Advancing VSVΔG-Env.BG505 toclinical trial will be valuable, as it will answer whether the livechimeric virus platform can be used to safely induce Env bindingantibodies with properties like those described above in healthyclinical trial volunteers.

Molecular Cloning, Recombinant Proteins, and Cell Line Development.

The VSV genomic clone is based on the VSV Indiana (IND) serotype (16).The plasmid vector containing the VSV genomic clone was similar to oneused before (46) except that the T7 RNA polymerase promoter was replacedwith a longer version that improves T7 RNA polymerase processivity(T7-g10 (47) and a hammerhead ribozyme sequence was positioned betweenthe T7-g10 promoter and the beginning of the VSV nucleotide sequence(48). The hepatitis delta virus ribozyme and T7 terminator sequencesdownstream of the 3′ end of the VSV antigenome were the same as usedbefore (46). Modified genomic clones with the G IND or G New Jersey (NJ)gene moved to genomic position 6 (VSV-G6, FIG. 19C) were describedearlier (16) and a third clone was developed for this study using Maraba(MAR) virus G (Genbank HQ660076.1). Plasmids that express the VSVstructural proteins (N, P, M, G, and L) under control of the CMVpromoter were used to support VSV rescue (16) instead of thosecontrolled by the T7 promoter used in the earlier procedure (46). Aplasmid designed to express T7 RNA polymerase from the CMV promoter wasdesigned similarly to the one described previously (46).

The Env immunogen expressed by VSVΔG-Env.BG505 and VSV-G6-Env.BG505 wasbased on the wild-type clade A Env.BG505 amino acid sequence (GenbankABA61516, 49, 50). Env.BG505 was modified by replacing the signalsequence, transmembrane region and cytoplasmic tail with correspondingregions of G from VSV IND (FIG. 23A). The nucleotide sequence encodingthe modified Env.BG505 was optimized with a VSV codon bias as describedpreviously (16) after which the gene was inserted in the VSV genomicclone in place of G. Additional VSVΔG-Env chimeras were developedsimilarly based on Env C.CH505 (week 100; Genbank KC247391.1) and EnvB.SF162p3 (Genbank KF042063).

A series of plasmids also were constructed to allow expression ofseveral different domains of Env.BG505 fused to the C-terminus of humanserum (HSA, 31). A glycine-serine linker (GGGGS(SEQ ID NO: 3)) wasinserted between the C-terminus of HSA and the Env sequence, and aC-terminal histidine tag was added to enable chromatographicpurification of HSA-fusion proteins secreted from transfected cells. TheHSA fusion proteins were expressed by transfecting 293T cells andpurified as described previously (49). His-tagged Env.BG505 gp120 (49)and gp140 containing a flexible linker in place of the furin cleavagesite (51) were expressed and purified similarly.

A stable VERO cell line expressing human CD4 and CCR5 (VERO-CD4/CCR5)was developed for propagating the VSVΔG-Env.BG505 vector. The human CD4and CCR5 coding sequences were joined by a 2A-like element (52) to forma single cistron (CCR5-2A-CD4), which was inserted into a plasmid underthe control of a transcription unit developed from the human heat shockprotein 60 gene (53). A stable cell line was generated by introducingDNA into cells by microporation (Neon Transfection System, Invitrogen)and selecting clonal isolates resistant to G418.

Cell Culture and Virus.

Recombinant virus recovery from DNA and virus propagation was performedusing VERO or VERO-CD4/CCR5 cells. Three media were used for VERO cellpropagation and electroporation procedures that were similar to thosedescribed before (46). VERO cell medium 1 (VCM1) is Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% heat-inactivated fetalbovine serum, 220 μM 2-mercaptoethanol, 2 mM L-glutamine, 1 mM sodiumpyruvate, and 0.1 mM MEM nonessential amino acids. VCM2 is Iscove'smodified Dulbecco's medium (IMDM) supplemented with 220 μM2-mercaptoethanol, 1% DMSO, 2 mM L-glutamine, 1 mM sodium pyruvate, and0.1 mM MEM nonessential amino acids. VCM3 is the same as VCM1 withaddition of 50 μg/mL Gentamicin. The VERO-CD4/CCR5 cell line waspropagated in VCM3 containing 1 mg per mL G418. All medium andsupplements were obtained from ThermoFisher.

Recombinant VSV was rescued from DNA using a helper-virus-free methodadapted from Witko et al. (46) using the modified plasmids describedabove. Virus rescue was initiated by electroporation of plasmidsencoding T7 RNA polymerase, VSV N, P, M, G, and L, and the appropriateVSV genomic clone into VERO (for VSV-G6) or VERO-CD4-CCR5 (for VSVΔG)cells. Conditions for electroporation with a BTX ECM 830 instrument(Harvard Apparatus) and subsequent virus recovery were similar to thoseused in the earlier method (46).

To ensure efficient vaccination with either vaccine, two vector-specificmodifications were applied, but the fundamental vaccine designs shown inFIGS. 19A-C were not changed. These modifications enhanced vaccinedelivery without altering the Env-dependent propagation ofVSVΔG-Env.BG505 or the G-dependent propagation of VSV-G6-Env.BG505. Themodifications are illustrated in FIGS. 23 and 24). To enhance mucosalVSVΔG-Env.BG505 uptake, vaccine material was prepared as a pseudotypedvirus particle bearing G (G pseudotype; FIGS. 23B and 24A-B). This wasdone simply by amplifying vaccine material in VERO-CD4/CCR5 cells thattransiently expressed VSV G. Pseudotyped VSVΔG-Env.BG505 launches a morerobust initial infection, because G recognizes a ubiquitous receptorfound on a wide range of cells (17). Importantly, G is not expressed bycells infected with pseudotyped VSVΔG-Env.BG505 and all subsequentrounds of infection in vivo are Env dependent (FIG. 23). ForVSV-G6-Env.BG505, it was modified to reduce the negative effects ofanti-G antibodies that develop during repeated vaccination with vectorsexpressing G. Three versions of VSV-G6-Env.BG505 (FIGS. 24A and C) wereused in sequence during the three-dose regimen (FIG. 24A). Each versionof VSV-G6-Env.BG505 differed only in the G gene (FIG. 24C), which wasexchanged with sequences from three different vesiculoviruses includingVSV IND, VSV NJ, and Maraba virus (16, 54).

Large batches of VSVΔG-Env.BG505 or VSV-G6-Env.BG505 were amplifiedusing VERO-CD4/CCR5 or VERO cells, respectively. Cell monolayers weregrown in Cell Factories (Corning) using VCM3, but once infection wasinitiated, the medium was changed to Virus Production Serum-Free Medium(VPSFM, supplemented with 4 mM L-Glutamine, 50 U/mL Penicillin and 50μg/mL Streptomycin; ThermoFisher). Cells were infected with ˜0.1plaque-forming units per cell and then incubated for about 24 h beforethe medium supernatant was harvested and clarified by centrifugation at900×g for 30 m at room temperature. Clarified supernatants were overlaidon 20% sucrose cushions prepared in phosphate-buffered saline (PBS),then centrifuged for 2 hrs (18,000 rpm, 42,900 g, 4° C.) using a SW28rotor (Beckman Coulter). The sucrose solution was aspirated completelyfrom the virus pellet after which virus was suspended in Hank's BalancedSalt Solution (HBSS, ThermoFisher) containing 15% Trehalose (LifeSciences Advanced Technologies) that was adjusted to pH 7.2. Virussuspensions were stored at −80° C. in aliquots.

Pseudotyped VSVΔG-Env.BG505 was produced in VERO-CD4/CCR5 cells thatwere electroporated with plasmid expressing VSV G IND or NJ. Inpreparation for electroporation, cells were harvested and treated asdescribed before (46) and were suspended in 0.7 ml of VCM2 (˜2×10⁷cells). Purified VSVΔG-Env.BG505 (0.1 pfu per cell) and 50 ug of pCMV-Gexpression plasmid was added to the cell suspension before performingelectroporation with the BTX ECM830 instrument. After electroporation,the cells were processed and transferred to one T175 flask perelectroporation cuvette, after which they were cultured in VCM1 for 3-4hours at 37° C. before performing heat shock (43° C.) for 3 hours (46).After heat shock, the cells were returned to 37° C. and allowed torecover for 2 h before the medium was removed and replaced VPSFMsupplemented with 4 mM L-Glutamine. Incubation was continued 24-48 hoursat 37° C. until cytopathic effect was evident throughout the cultureafter which virus was harvested and purified as described above.

VSV vector infectious units were quantified by plaque assay (16). ForVSVΔG-Env.BG505, GHOST-CD4-CCR5 cell monolayers were used (NIH AIDSReagent Program, Division of AIDS, NIAID, NIH, catalog number 3944, 55)while VERO cells were used for VSV-G6-Env.BG505. Near-confluent cellmonolayers were infected with serially diluted virus before beingoverlaid with VCM3 containing 0.8% agarose. When plaques were visible,cells were fixed with 7% formaldehyde and stained with a solution of 7%crystal violet in water. Plaques were counted from duplicate wells andinfectious titers were expressed as plaque-forming units (pfus) per ml.

Western blotting was used to confirm Env expression by infected cellsand also to characterize purified VSV vector particles. For analysis ofEnv expression, cytoplasmic lysates were prepared from infectedmonolayers using CellLytic M reagent (Sigma). Lysate proteins were thensubjected to denaturing SDS polyacrylamide gel electrophoresis (SDSPAGE) and transferred to nitrocellulose membranes. Proteins weredetected with monoclonal antibodies or polyclonal serum specific for Envgp120.BG505 or VSV structural proteins. Secondary antibodies conjugatedto horse-radish peroxidase and chemiluminescence detection was used tovisualize specific bands. Protein composition of VSV vector particleswas analyzed by Western blot using similar methods applied to viruspurified by centrifugation through sucrose cushions.

VSV vector vaccine material was subjected to several tests to ensure thequality. Endotoxin levels were tested using the Endosafe Portable TestSystem (Charles River Laboratories, Boston). All vaccine lots hadendotoxin levels less than 10 EU/ml. The absence of Mycoplasma wasconfirmed by PCR using the MycoSEQ® Mycoplasma Detection System (LifeTechnologies). Residual VERO cell DNA was less than 10 ng per dose asdetermined with the resDNASEQ® Vero Residual DNA Quantitation System(Life Technologies). Gene sequences were confirmed by nucleic acidsequencing as described before (16).

Vaccinations, SHIV Challenge, and Animal Care and Use.

Purpose-bred male Indian rhesus macaques were 4-7 years of age when theyarrived at The State University of New York (SUNY) Downstate MedicalCenter, Division of Comparative Medicine. Animal care and use compliedwith The United States Department of Agriculture and The New York StateDepartment of Health regulations. The SUNY Downstate Medical CenterInstitutional Animal Care and Use Committee reviewed all experimentalprocedures. Prior to receipt, all macaques were confirmed to be negativefor Herpes B virus (BV), tuberculosis (TB), simian immunodeficiencyvirus (SIV), simian retrovirus (SRV), and simian T lymphotropic virus(STLV), as well as Shigella and Campylobacter jejuni.

No Macaques were included in the study if they were positive for MHCalleles Mamu-B*08 and B*17 associated with strong SIV replicationcontrol (56). Both groups vaccinated with VSV vectors each had 2 animalsthat were positive for Mamu-A*01 and two positive A*02, which have beenassociated with control of disease progression (56). The placebo controlgroup also contained two animals that were positive for A*02 and one forA*01. For vaccination, macaques were sedated and positioned in dorsalrecumbency after which vaccine was administered by the intraoral andintranasal routes. Vaccine or buffer control was administered by dropsusing a 1000 μl micropipette. 500 μl was delivered intranasally byalternating drops between the left and right nares, with time betweendrops allowed for the droplet to be inhaled. For intraoral, a total of500 μl was administered by drops applied sublingually on the frenulum(250 μl) and to the anterior buccal surface of the inferior lip (250 μl)followed by 30-60 seconds of gentle massage to help distribute theinoculum. Animals were kept in dorsal recumbency throughout theprocedure and were left in this position for an additional 5 minutesbefore being returned to their cages. Animals were singly housed for 48h following all vaccinations, after which they were housed together (2-3animals per cage) within the same vaccination group. Bedding materialwas analyzed for VSV genomes by qPCR and none was detected (data notshown).

Rectal challenge was performed using SHIV SF162p3 that was prepared inprimary cultures of macaque PBMCs (34). The inoculum (total of 2.2×10⁴TCID50) consisted of virus in 1 ml of saline or RPMI medium. Sedatedanimals were positioned in sternal recumbency with the posteriorelevated by placing an empty plastic container between the lower abdomenand the procedure table. Inoculation was performed by atraumaticinsertion of a lightly lubricated 3 mL syringe approximately 5 cm intothe rectum. The inoculum was slowly instilled over a one-minute periodwith the syringe left in place for and additional 4 minutes. Afterremoving the syringe, macaques remained in sternal recumbency for 10minutes. Challenged animals were caged separately for 48 h before beinghoused in groups of 2-3 within the same vaccination group.

SHIV infection was monitored by reverse transcription and quantitativePCR (RT-qPCR) using methods similar to those described earlier (57).Briefly, virus from 1.0 ml of plasma was collected by centrifugation at25,000×g for 90 min (5° C.). The virus pellet was processed using theRNeasy Micro kit (Qiagen) by suspending virus in solution containing 300μl of lysis buffer, 3 μl of 14.2 M 2-mercaptoethanol (Bio-Rad), and 16μl of 20 mg/ml proteinase K (Qiagen). Samples were digested at 56° C.for 1 h, then RNA was purified using spin columns following the RNeasyMicro kit protocol. RNA was eluted in 50 μl of RNase-free watersupplemented with 1 mM dithiothreitol (Sigma) and 1 U/μl RNAseOUT(Thermo Fisher Scientific) after which duplicate RT reactions wereperformed using 15 μl of purified RNA per reaction and 10 μl of acocktail composed of reagents from the Sensiscript Reverse Transcriptasekit (Qiagen, Valencia, Calif., USA) including 1× reverse transcriptionbuffer, 0.5 mM of each dNTP, 10 U/reaction RNase Inhibitor (Invitrogen,Carlsbad, Calif., USA), 10 Units Sensiscript Reverse transcriptase, andGag-specific reverse primer (400 nM,5′-CACTAGKTGTCTCTGCACTATPTGTTT-3′(SEQ ID NO: 4)) that annealed to thepositive-sense genomic RNA. Reverse transcription was performed at 50°C. for 45 min and terminated by heat inactivation (95° C. for 2 min).The heat-inactivated 25-μl reaction was adjusted for qPCR by adding 30μl of a reagent mix composed of 1× QuantiTect Multiplex PCR Master Mix(Qiagen), 400 nM of Gag-specific forward primer(5′-GTCTGCGTCATPTGGTGCAT-3′ (SEQ ID NO: 5)) and Gag-specific reverseprimer, and 200 nM 6-carboxyfluorescein (FAM)-labeled minor groovebinder (MGB) probe (5′-6FAM-CTTCPTCAGTKTGTTTCA-MGB-3′ (SEQ ID NO: 6)). AStratagene Mx3005P Sequence Detection System was used for amplificationand detection with the following conditions: 15 min at 95° C. followedby 45 cycles of 60 secs at 94° C. and 90 secs at 60° C. Results fromduplicate test samples were averaged and genome copy numbers wereinterpolated from a curve generated with known RNA standards. Positivesamples were defined as 200 genome copies per ml of plasma.

Analysis of Immune Responses.

To prepare plasma and peripheral blood mononuclear cells (PBMCs), bloodwas collected in tubes coated with sodium heparin. Plasma was preparedby removing cells by centrifugation and storage at −20° C. PBMCs wereisolated by density gradient centrifugation on Ficoll Hypaque (GEHealthcare) in Accuspin tubes (Sigma-Aldrich) as described previously(57). Harvested PBMCs were suspended in Recovery Cell Culture FreezingMedium (Thermo Fisher Scientific) and stored in liquid nitrogen. Serumused for ELISA, Western blot procedures, binding antibody multiplexassays, or HIV pseudovirus neutralization assays was prepared from wholeblood collected and processed in serum separator tubes (SST). Aliquotswere stored at −20° C.

Intracellular cytokine staining was performed as described before (57).T cells were stimulated with Env.BG505 peptide (Genscript) 15-mersoverlapping by 11 amino acids. Two different Env.BG505 peptide pools,spanning gp120 or gp41, were used at 4 μg per ml. All flow cytometrydata had mock background responses subtracted.

Infected VERO and VERO-CD4/CCR5 cells and VSV vector particles also wereanalyzed by flow cytometry. For infected cells, VERO or VERO-CD4/CCR5monolayers were infected with 0.1 to 1.0 pfu per cell and incubatedovernight at 37°. The following day, cells were washed with PBS and thentreated with Enzyme-free Cell Dissociation Buffer (Life Sciences) toproduce a cell suspension. The cells were collected by centrifugationand then suspended in PBS before being incubated with Env-specificmonoclonal antibodies. Flow cytometry was performed as described earlier(16).

Env incorporated in VSV particles also was analyzed by flow cytometry(16). Typically, purified virus (10⁸ pfus) was bound to 100 ug Alum(Adju-Phos, Brenntag, Denmark) and the alum/virus complexes were blockedwith PBS containing 3% BSA before being incubated with primaryantibodies. After primary antibody incubation, the complexes werecollected by centrifugation, washed using PBS containing 3% BSA, andthen incubated with labeled secondary antibody. Centrifugation andwashing was repeated before analysis with a LSRII flow cytometer (BectonDickinson). The flow cytometer was set to analyze 30,000 particles withforward scatter (FSC) and side scatter (SSC) set to log 10 scale andthreshold set to 4000. Data was analyzed using FlowJo software version9.4 (Tree Star), where complexes were gated according to positivitycompared to an alum only control.

Western blotting also was used for analysis of serum antibodies.Polypeptide substrates used for the analysis were either purifiedVSVΔG-EnvG.BG505 particles (no G pseudotype, 5×10⁸ pfus) or purified Envproteins. Purified virus or protein was diluted to 162.5 μL in HBSScontaining 15% Trehalose before being mixed with 62.5 μl LDS NuPAGEsample buffer (Novex) and 25 μL of NuPAGE Sample Reducing Agent (Novex).Samples were heat denatured before being electrophoresed in a denaturingpreparative gel (NuPAGE 4-12% Bis-Tris 2D, ThermoFisher), and afterwardsproteins were transferred to a nitrocellulose membrane. The membrane wasrinsed with PBS and then incubated at room temperature for 1 h inblocking buffer composed of StartingBlock T20 buffer (ThermoFisher)supplemented with Clear Milk (Pierce/ThermoFisher) and 1% goat serum(Sigma). The blocked membrane was transferred to a multichannel MiniProtein II MultiScreen (BioRad) device that created multiple channelsfor analysis of sera from individual macaques. Individual lanes wereincubated for 1 h at room temperature with heat-inactivated macaqueserum (diluted 1:300 in blocking buffer for a total volume of 550 μL)before the solution was aspirated completely from each lane. Themembrane was then removed from the multiscreen device and rinsed 5 timeswith miliQ water (59) and then washed 3 times for 5 minutes each withPBS containing 0.1% Tween-20. The membrane was incubated with secondaryantibody (mouse anti-monkey IgG, SouthernBiotech; diluted 1:10,000 inblocking buffer) for 45 mins at room temperature after which it waswashed as described above. The blot was developed with chemiluminescencereagent (SuperSignal West Femto Maximum Sensitivity Substrate,ThermoFisher) and imaged with a Biorad ChemiDoc Touch Imaging System.

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Example 3: Using VSV Evolution to Fine Tune the Env Immunogen

The EnvG hybrid was developed with the goal of producing an immunogenthat was optimal for delivery with a live VSV vector. The Env-G designobjectives were to generate a modified immunogen that enabled moreabundant expression on the infected cell surface and increasedincorporation in VSV particles while maintaining native Env antigenicproperties and the ability to direct infection and replication in cellsbearing the HIV coreceptors CD4 and CCR5 (CD4+/CCR5+ cells). Systematicevaluation of several Env domain substitutions demonstrated thatreplacement of the Env signal peptide (SP), transmembrane region (TM),and cytoplasmic tail (CT) with analogous domains from VSV Gsubstantially improved expression of Env on the cell surface (FIG. 1).Moreover, the surface of cells expressing the Env-G hybrid was bound bya panel of anti-Env monoclonal antibodies demonstrating that theantigenic profile was very similar to Env expressed by cells infectedwith HIV. Importantly, when a chimeric VSVΔG-Env vector was developed inwhich the G gene was replaced with Env-G, replication-competentrecombinant virus was isolated that replicated specifically inCD4+/CCR5+ cells demonstrating that the EnvG retained functions that areessential for cell attachment and virus replication.

The domain swap approach enabled development of a live VSVΔG-Env chimerathat readily propagated in CD4+/CCR5+ cells. After conducting multiplerounds of amplification in CD4+/CCR5+ cells, virus emerged that grew tohigher titers suggesting that one or more mutations occurred resultingin a virus with increased replicative fitness. Genomic sequence analysisconducted on this virus strain identified three amino acid substitutionsin Env, which were (amino acid numbering according to reference strainHXB2): K169T in the second variable domain of Env (V2 domain), I307T inthe V3 domain, and W672R in the membrane-proximal external region(MPER). Consistent with these substitutions being the adaptive mutationsthat improve replicative fitness of the virus, the three amino acidchanges have been stable during numerous subsequent rounds of viruspropagation. Moreover, there was a substantial difference in virusquantities produced from infected cultures; VSVΔG-Env.BG505 with thethree substitutions routinely exceeds 1×10e7 PFU per ml of medium whileVSVΔG-Env.BG505 amplified prior to adaptation produced titers closer to1×10e6.

To provide additional evidence that the three substitutions were theresult of adaptive mutations that improved replicative fitness, themutations were incorporated into the VSVΔG-Env.BG505 genomic DNA cloneand a new recombinant virus was recovered containing the Envsubstitutions. This new recombinant strain grew efficiently, maintainedthe three amino acid substitutions during many rounds of propagation,and accrued no additional EnvG mutations. These results indicated thatthe three amino acid substitutions provided a replicative fitnessadvantage for the VSVΔG-Env.BG505 chimera.

The accrual of the three amino acid substitutions that enhancedreplicate fitness indicated that EnvG structure likely required someadditional ‘fine tuning’ to support optimal VSVΔG-Env.BG505 growth. Thesubstitutions probably compensated for some structural changes in theEnv complex that resulted from replacement of TM and CT with VSV Gsequence. Structural changes in the Env complex are known to occur whenmutations are introduced into the Env TM (1) and CT (2); thus, it isreasonable to expect that replacement of the Env TM and CT with VSV Gsequence will cause some structural alteration that requirescompensatory second-site mutations to achieve optimal EnvG function andvirus replicative fitness.

It was noticeable that the three Env substitutions occurred in the Envectodomain rather than in the G TM or CT. This probably reflects strongselective pressure to maintain the wild-type G TM and CT sequence, asthey are optimal for VSV particle structure, and in fact, the G CT makescontact with the underlying VSV matrix protein (3). Thus, selectivepressure favored accrual of compensatory amino acid changes in the Envectodomain rather than in the G TM or CT.

It was also notable that the adaptive mutations occurred in threeseparate regions of the Env ectodomain including the gp120 (V2 K169T andV3 I307T) and gp41 (MPER W672R) subunits. The mechanism by which thiscombination of amino acid substitutions improves replicative fitness isunknown. Furthermore, this makes it difficult to predict whatsubstitutions might be useful for optimizing propagation of a chimericvirus like VSVΔG-EnvG.BG505; thus, VSV's ability to rapidly evolve whenfaced with selective pressure (4) is an important tool in the overallVSVΔG-Env vaccine design process.

To demonstrate the importance of VSV evolution in design of an optimalimmunogen and chimeric virus vector, an independent VSVΔG-Env.BG505recombinant was isolated that lacked adaptive mutations and it wasallowed to evolved during serial rounds of propagation. The resultsshowed that the virus did in fact accrue multiple amino acidsubstitutions as before, but the constellation of adaptive mutations wasdifferent. After multiple rounds of amplification, this new strain had 4substitutions (Table). Interestingly, as before, one of thesubstitutions was in V2 (E164G). The other three were in constant (C)domains of Env (C4 M434T, C4 Q440R, and C5 L494F).

The VSVΔG-Env.BG505 vaccine containing the K169T, I307T and W672R wasfound to be efficacious in the Indian Rhesus macaque SHIV challengemodel.

CITATIONS

-   1. Lovelace E, Xu H, Blish C A, Strong R, Overbaugh J. The role of    amino acid changes in the human immunodeficiency virus type 1    transmembrane domain in antibody binding and neutralization.    Virology. 2011; 421(2): 235-44.-   2. Chen J, Kovacs J M, Peng H, Rits-Volloch S, Lu J, Park D, et al.    HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic    characteristics of HIV-1 envelope glycoprotein. Science. 2015;    349(6244):191-5.-   3. Ge P, Tsao J, Schein S, Green T J, Luo M, Zhou Z H. Cryo-EM model    of the bullet-shaped vesicular stomatitis virus. Science. 2010;    327(5966):689-93.-   4. Novella I S. Contributions of vesicular stomatitis virus to the    understanding of RNA virus evolution. Curr Opin Microbiol. 2003;    6(4):399-405.

Example 4: Transgenic Vero-CD4/CCR5 Cell Line

Human immunodeficiency virus (HIV) or its simian equivalent simianimmunodeficiency virus (SIV) initiates infection through the viralenvelope (Env) membrane glycoprotein binding to cellular CD4 and acoreceptor such as CCR5. Similarly, Env pseudotyped viral vectors canalso infect cells by recognizing the coreceptors. In vitro, HIV, SIV,and Env pseudotyped replicating viral vectors can be propagated onprimate peripheral blood mononuclear cells (PBMCs) or transgenic celllines that are engineered to constitutively express CD4/CCR5. To supportpropagation and production of chimeric vesicular stomatitis virus (VSV)and canine distemper virus (CDV) vectors that have Env in place ofnative vector membrane glycoproteins, Applicants generated a transgenicVero cell lines that express either human or rhesus macaque CD4/CCR5.Optimized transgene design and transfection procedures make transgenicVero-CD4/CCR5 derivation process efficient and reliable. Stable cloneswere selected through rigorous multiple rounds of limiting dilutions.The VSV- and CDV-Env chimeras grow efficiently on the transgenicVero-CD4/CCR5 cell lines and express Env of native conformation andantigenicity. Because Vero is a FDA-approved cell substrate for humanvaccine production, the transgenic Vero-CD4/CCR5 cells have substantialpotential to be used for manufacturing of replicating viral vectored HIVvaccines that express functional Env immunogens.

Most CD4/CCR5 expressing transgenic cell lines (e.g. GHOST, HOS, A3R5,and TZM-bl) were for use in analytical assays, but are not suitable forHIV vaccine manufacturing. The Vero cell line is approved for producinghuman vaccines (e.g. inactivated polio vaccine), therefore transgenicVero-CD4/CCR5 cells are useful for HIV vaccine production since manysafety risks associated with cell substrates have been addressed for theVero cell background. In addition, the unique CD4/CCR5 transgene designdirects strong expression of a CCR5 and CD4 polyprotein linked by a 2Asequence that is subsequently self-cleaved resulting in 1 to 1 ratio ofCD4 and CCR5 molecules.

The transgenic Vero-CD4/CCR5 cell lines can be used for producingreplicating viral vectors expressing HIV or SIV Env. Their use can alsobe expanded for use in assays requiring cells expressing CD4 and CCR5.

As the expression cassette proved effective with CD4 and CCR5, it isuseful for making cell lines expressing other polypeptides.

FIGS. 33A-33E. Sequence annotation of VERO-hCD4/CCR5 gene. A restrictionmap of the Vert construct shows restriction enzymes cutting a maximum oftwo times, using RELibrary as a restriction enzyme library.

FIG. 34. Gene design: VERO-CD4/CCR5 Cell Line (VERT). VERO-hCD4/CCR5:Transgenic Vero cells expression human CD4 and CCR5 receptors. sVERT3:Transgenic Vero cells expression simian CD4 and CCR5 receptors.

FIG. 35. VERO-hCD4/CCR5 clone 4F11 resembles the Vero cell.

FIG. 36. VERO-hCD4/CCR5 cytopathic effect when infected by VSV chimera.

FIG. 37. VERO-hCD4/CCR5 maintains infectivity over 20 passages.

FIG. 38. VERO-hCD4/CCR5 maintains the receptor expression over 20passages.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A Vero cell line transformed with and expressinga cluster of differentiation 4 (CD4) receptor and a C—C chemokinereceptor type 5 (CCR5 receptor).
 2. The cell line of claim 1, whereinthe CD4 receptor and CCR5 receptor is a human CD4 receptor and a humanCCR5 receptor.
 3. The cell line of claim 1, wherein the CD4 receptor andCCR5 receptor is a simian CD4 receptor and a simian CCR5 receptor.
 4. Anucleic acid comprising the construct of FIG.
 33. 5. The cell line ofclaim 1, wherein the cell line is transformed with the construct of FIG.34.
 6. The cell line of claim 1, wherein the cell line is transformedwith the construct of FIG.
 35. 7. The cell line of claim 1 transfectedwith a recombinant vesicular stomatitis virus (VSV) vector wherein agene encoding the VSV surface glycoprotein G (VSV G) is functionallyreplaced by functional clade A HIV Env BG505.
 8. The cell line of claim1 transfected with a vector containing and expressing a nucleic acidsequence encoding an amino acid sequence of an Env.BG505 immunogenencoded by the VSVΔG-Env.BG505.
 9. The cell line of claim 8, wherein theamino acid sequence of an Env.BG505 immunogen encoded by theVSVΔG-Env.BG505 is SEQ ID NO:
 2. 10. The cell line of claim 8 whereinthe vector comprises the sequence of a VSVΔG-Env.BG505 genomic clone.11. The cell line of claim 10, wherein the sequence of a VSVΔG-Env.BG505genomic clone is SEQ ID NO: 1.12. A method for propagating a VSV or CDVvaccine vector wherein a VSV or CDV envelope is replaced with a HIV orSIV envelope protein comprising transfecting the cell line of any one ofclaims 1 to 3 with the VSV or CDV vaccine vector.